U.S. patent number 9,095,589 [Application Number 13/444,642] was granted by the patent office on 2015-08-04 for chirally pure isomers of itraconazole for use as angiogenesis inhibitors.
This patent grant is currently assigned to Johns Hopkins University. The grantee listed for this patent is Shridhar Bhat, Curtis R. Chong, Jun O. Liu, Jun Lu, Wie Shi, Jing Xu. Invention is credited to Shridhar Bhat, Curtis R. Chong, Jun O. Liu, Jun Lu, Wie Shi, Jing Xu.
United States Patent |
9,095,589 |
Liu , et al. |
August 4, 2015 |
Chirally pure isomers of itraconazole for use as angiogenesis
inhibitors
Abstract
Described herein are methods of inhibiting angiogenesis, and
treating and preventing disorders associated with angiogenesis by
administering anti-angiogenesis compounds to a subject.
Inventors: |
Liu; Jun O. (Clarkesville,
MD), Chong; Curtis R. (Honolulu, HI), Xu; Jing
(Parkville, MD), Lu; Jun (Monmouth JCT, NJ), Bhat;
Shridhar (Cockeysville, MD), Shi; Wie (Fayetville,
AR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Liu; Jun O.
Chong; Curtis R.
Xu; Jing
Lu; Jun
Bhat; Shridhar
Shi; Wie |
Clarkesville
Honolulu
Parkville
Monmouth JCT
Cockeysville
Fayetville |
MD
HI
MD
NJ
MD
AR |
US
US
US
US
US
US |
|
|
Assignee: |
Johns Hopkins University
(Baltimore, MD)
|
Family
ID: |
48136460 |
Appl.
No.: |
13/444,642 |
Filed: |
April 11, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130102614 A1 |
Apr 25, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12594777 |
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PCT/US2008/004513 |
Apr 7, 2008 |
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60922059 |
Apr 5, 2007 |
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61474052 |
Apr 11, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/4178 (20130101); A61K 31/496 (20130101); A61K
45/06 (20130101); A61K 31/4178 (20130101); A61K
2300/00 (20130101); A61K 31/496 (20130101); A61K
2300/00 (20130101); Y02A 50/401 (20180101); Y02A
50/30 (20180101) |
Current International
Class: |
A61K
31/496 (20060101); A61K 31/4178 (20060101); A61K
45/06 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
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4267179 |
May 1981 |
Heeres et al. |
4616027 |
October 1986 |
Richardson et al. |
5474997 |
December 1995 |
Gray et al. |
6166018 |
December 2000 |
McCullough et al. |
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Foreign Patent Documents
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WO/2006/004795 |
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Dec 2006 |
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WO |
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WO/2008/124132 |
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Oct 2008 |
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WO |
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Other References
Baker et al. Stereochemistry and drug efficacy and development:
relevance of chirality to antidepressant and antipsychotic drugs,
Annals of Medicine (Stockholm, Sweden), 2002, 24 (7/8), 537-543.
cited by examiner .
Chong, C. R. et al . , `Inhibition of angiogenesis by the
antifungal drug itraconazole`, ACS Chemical Biology, 2007, vol. 2,
No. 4, pp. 263-270. cited by applicant .
International Search Report regarding PCT/US2013/036024, mailed:
Jul. 26, 2013. cited by applicant .
Zarn et al., "Azole Fungicides Affect Mammalian Steroidogenesis by
Inhibiting Sterol 14.alpha. Demethylase and Aromatase",
Environmental Health Perspectives, 111(3):255-261 (2003). cited by
applicant .
Shi et al., "Impact of Absolute Stereochemistry on the
Antiangiogenic and Antifungal Activities of Intraconzale", ACS Med
Chem Lett 1(4):155-159 (2010). cited by applicant.
|
Primary Examiner: Justice; Gina
Assistant Examiner: Alley; Genevieve S
Attorney, Agent or Firm: DLA Piper LLP (US)
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION(S)
This application is a continuation-in-part of U.S. Ser. No.
12/594,777, filed on May 2, 2011, now pending, which is a 35 U.S.C.
.sctn.371 National Stage Application of PCT Application No.
PCT/US08/04513 filed Apr. 7, 2008, now abandoned; which claims the
benefit under 35 U.S.C. .sctn.119(e) to U.S. Application Ser. No.
60/922,059, filed Apr. 5, 2007, now abandoned; and claims the
benefit of priority under 35 U.S.C. .sctn.119(e) of U.S. Ser. No.
61/474,052 filed Apr. 11, 2011. The disclosure of each of the prior
applications is considered part of, and is incorporated by
reference in, the disclosure of this application.
Claims
What is claimed is:
1. A method of inhibiting angiogenesis in a subject, comprising
administering to the subject an effective amount of a chirally pure
compound of structural Formula A, wherein the compound is
anti-angiogenic: ##STR00036##
2. The method of claim 1, wherein the compound is administered at a
dose of a dosage of between about 0.1 and 100 mg/kg/day.
3. The method of claim 1, wherein the administering of the
anti-angiogenic compound comprises administering the compound in a
dosage of less than about 500 mg/day.
4. The method of claim 1 comprising the step of administering an
effective amount of a composition comprising the anti-angiogenic
compound and a pharmaceutically suitable excipient.
5. The method of claim 1, wherein the administering of the
anti-angiogenic compound comprises administering the compound
orally, topically, parentally, intravenously or
intramuscularly.
6. The method of claim 5, wherein the administration is carried out
in a controlled and sustained release.
7. The method of claim 1, wherein the subject is a human.
8. The method of claim 1, wherein the compound is administered in
an amount effective for treatment of retinoblastoma, cystoid
macular edema (CME), macular degeneration, exudative age-related
macular degeneration (AMD), diabetic retinopathy, diabetic macular
edema, or ocular inflammatory disorders.
9. The method of claim 1, wherein the compound is administered in
an amount effective for treatment of a disease or disorder
associated with a tumor or cancer of the breast.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to angiogenesis and, more
specifically, to compounds and compositions for the treatment of
disorders associated with angiogenesis.
2. Background Information
Angiogenesis may be defined as the development of a blood supply to
a given area of tissue. The development of a blood supply may be
part of normal embryonic development, represent the
revascularization of a wound bed, or involve the stimulation of
vessel growth by inflammatory or malignant cells. Sometimes
angiogenesis is defined as the proliferation of new capillaries
from pre-existing blood vessels. New growth of soft tissue requires
new vascularization, and the concept of angiogenesis is a key
component of tissue growth and in particular, a key point of
intervention in pathological tissue growth.
Angiogenesis is a fundamental process necessary for embryonic
development, subsequent growth, and tissue repair. Angiogenesis is
a prerequisite for the development and differentiation of the
vascular tree, as well as for a wide variety of fundamental
physiological processes including embryogenesis, somatic growth,
tissue and organ repair and regeneration, cyclical growth of the
corpus luteum and endometrium, and development and differentiation
of the nervous system. In the female reproductive system,
angiogenesis occurs in the follicle during its development, in the
corpus luteum following ovulation and in the placenta to establish
and maintain pregnancy. Angiogenesis additionally occurs as part of
the body's repair processes, e.g., in the healing of wounds and
fractures.
Nevertheless, angiogenesis is implicated in a number of important
human diseases including cancer, diabetic retinopathy, and
rheumatoid arthritis. Since the angiogenesis hypothesis was first
put forward in 1971, the physiologic and pathological roles of
angiogenesis in various biological and disease processes have been
subject to extensive scrutiny. The importance of angiogenesis in
human diseases such as cancer is well established. Significant
progress in anti-angiogenic drug discovery and development has also
been made, culminating in the development of angiogenesis
inhibitors as drugs for the treatment of cancer and age-related
macular degeneration. Angiogenesis inhibitors have been found to be
particularly useful when used in conjunction with other
chemotherapeutic drugs. Angiogenesis also contributes to the
pathogenesis of a number of other diseases, including obesity,
psoriasis, Kaposi's sarcoma, diabetic retinopathy, pulmonary
hypertension, and arthritis. It is thus not surprising that an
estimated 500 million people worldwide may benefit from treatments
that modulate angiogenesis.
A number of existing drugs have been found to possess
anti-angiogenic effects either serendipitously or by rational
prediction. One of the first anti-angiogenic drug candidates to
enter clinical trials is TNP-470, a derivative of the anti-amebic
drug fumagillin, which was discovered in the late 1980s from a
fungal contamination that inhibited endothelial cell culture
growth. Other existing drugs such as thalidomide, non-steroidal
anti-inflammatory agents and rapamycin also inhibit angiogenesis
and have shown promise in clinical trials for the treatment of
cancer. Although new uses for several dozen existing drugs such as
fumagillin have been found serendipitously or through knowledge of
pharmaceutical side effects, a systematic assembly and screening of
libraries of existing drugs for novel pharmacological activities
did not begin until recently. Consequently, there is a need for new
specific targets which can be indicative for angiogenesis
inhibition. It is therefore an object of the invention to provide a
target for biological screening of compounds for angiogenesis
inhibition.
Furthermore, many materials which appear promising in vitro have
proven to be relatively ineffective when applied in vivo.
Similarly, various of such materials have been found to be
unstable, toxic, or otherwise difficult to employ. Consequently,
there is a need for methods and materials capable of controlling
and inhibiting angiogenesis in a reliable manner. It is therefore
an object of the invention to provide compounds and pharmaceutical
compositions which exhibit activity as inhibitors of
angiogenesis.
SUMMARY OF THE INVENTION
The present invention is based on the seminal discovery that the
stereochemistry at one end of itraconazole appears to have an
influence on its biological activity. The anti-fungal activity of
itraconazole is influenced by stereochemistry with trans
stereoisomers demonstrating potency on the same order of magnitude
as cis stereoisomers.
In one aspect, the invention provides a method for identifying a
compound useful for the inhibition of angiogenesis or the treatment
of a disease or disorder associated with angiogenesis, comprising
the step of determining the lanosterol 14.alpha.-demethylase
inhibitory activity of said compound.
In another aspect, the invention provides a method of inhibiting
angiogenesis in a subject, the method comprising the step of
administering to the subject an effective amount of an inhibitor of
lanosterol 14.alpha.-demethylase.
In another aspect, the invention provides a method of inhibiting
angiogenesis in a subject, the method comprising the step of
administering to the subject an effective amount of chirally pure
4S-cis-itraconazole or a chirally pure compound of structural
Formulas A-H:
##STR00001## ##STR00002##
In another aspect, the invention provides a method of inhibiting
angiogenesis in a subject, the method comprising the step of
administering to the subject an effective amount of chirally pure
4R-cis-itraconazole or a chirally pure compound of structural
Formulas A-H. In certain aspects, the compound administered is a
chirally pure compound of structural Formulas A or B. In other
aspects, the compound administered is a chirally pure compound of
structural Formulas C or D.
In another aspect, the invention provides a method of inhibiting
angiogenesis in a subject, the method comprising the step of
administering to the subject an effective amount of azalanstat.
In another aspect, the invention provides the use of an
anti-angiogenic compound in the manufacture of a medicament for
inhibiting or reducing angiogenesis in a patient, where the
anti-angiogenic compound is, for example, chirally pure
4S-cis-itraconazole, chirally pure 4R-cis-itraconazole, azalanstat,
an inhibitor of lanosterol 14.alpha.-demethylase, or a chirally
pure compound of structural Formulas A-H.
In yet another aspect, the invention provides a sustained release
device for implantation in a patient and sustained release of an
anti-angiogenic compound for at least a period of 30 days, wherein
the anti-angiogenic compound is, for example, chirally pure
4S-cis-itraconazole, chirally pure 4R-cis-itraconazole, azalanstat,
an inhibitor of lanosterol 14.alpha.-demethylase, or a chirally
pure compound of structural Formulas A-H.
In yet another aspect, the invention provides a sustained release
drug device adapted for implantation in or adjacent to the eye of a
patient, the drug delivery device comprising: (i) a drug core
comprising anti-angiogenic compound being: chirally pure
4S-cis-itraconazole, chirally pure 4R-cis-itraconazole, azalanstat,
an inhibitor of lanosterol 14.alpha.-demethylase, or a chirally
pure compound of structural Formulas A-H; (ii) an impermeable
coating disposed about the core that is substantially impermeable
to the passage of the anti-angiogenic compound, having one or more
openings therein which permit diffusion of the anti-angiogenic
compound, and which is substantially insoluble and inert in body
fluids and compatible with body tissues; and, optionally, (iii) one
or more permeable polymer members or coatings disposed in the flow
path of the anti-angiogenic compound through said openings in said
impermeable coating, said permeable polymer being permeable to the
passage of the anti-angiogenic compound, and which is substantially
insoluble and inert in body fluids and compatible with body
tissues; wherein the impermeable coating and permeable polymer
members or coatings are disposed about the drug core so as to
produce, when implanted, a substantially constant rate of release
of the anti-angiogenic compound from the device.
In another aspect, the invention provides a sustained release
formulation for depot injection in a patient and sustained release
of an anti-angiogenic compound for at least a period of 30 days,
wherein the formulation includes:
a viscous gel formulation comprising a bioerodible, biocompatible,
polymer; and
an anti-angiogenic agent dissolved or dispersed therein, which
anti-angiogenic agent is: chirally pure 4S-cis-itraconazole,
chirally pure 4R-cis-itraconazole, azalanstat, an inhibitor of
lanosterol 14.alpha.-demethylase, or a chirally pure compound of
structural Formulas A-H.
In one embodiment, the invention provides a method, use, device, or
formulation, wherein the anti-angiogenic compound is provided in an
amount effective for treatment of retinoblastoma, cystoid macular
edema (CME), exudative age-related macular degeneration (AMD),
diabetic retinopathy, diabetic macular edema, or ocular
inflammatory disorders.
In another embodiment, the invention provides a method, use,
device, or formulation for treatment of a tumor.
In another embodiment, the invention provides a method, use,
device, or formulation for the treatment of dermis, epidermis,
endometrium, retina, surgical wound, gastrointestinal tract,
umbilical cord, liver, kidney, reproductive system, lymphoid
system, central nervous system, breast tissue, urinary tract,
circulatory system, bone, muscle, or respiratory tract.
In yet another embodiment, the invention provides a method, use,
device, or formulation for eliminating or reducing normal but
undesired tissue in a patient.
In still another embodiment, the invention provides a method, use,
device, or formulation for the reduction of fat.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the anti-angiogenic compound
inhibits endothelial cell proliferation.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the anti-angiogenic compound
inhibits G1/S cell cycle progression of endothelial cells.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the anti-angiogenic compound
decreases new blood vessel formation.
In another embodiment, the invention provides a method, use, device
or formulation, further comprising an additional therapeutic
agent.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the additional therapeutic agent is
an angiogenesis-inhibiting compound. In a certain embodiment, the
invention provides a method, use, device or formulation, wherein
the additional therapeutic agent is an anticancer compound.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the step of administering the
anti-angiogenic compound comprises administering the compound
orally, topically, parentally, intravenously or
intramuscularly.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the administration is carried out in
a controlled and sustained release.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the step of administering the
anti-angiogenic compound comprises administering the compound in a
dosage of between about 0.1 and 100 mg/kg/day.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the step of administering the
anti-angiogenic compound comprises administering the compound in a
dosage of less than about 500 mg/day.
In a certain embodiment, the invention provides a method, use,
device or formulation, wherein the subject is a human.
In another aspect, the invention provides a kit comprising an
effective amount of an anti-angiogenic compound in unit dosage
form, together with instructions for administering the
anti-angiogenic compound to a subject suffering from or susceptible
to a disease or disorder or symptoms thereof associated with
angiogenesis.
In a certain embodiment, the invention provides a method, use,
device or formulation, comprising the step of administering an
effective amount of a composition comprising an anti-angiogenic
compound and a pharmaceutically suitable excipient.
In a certain embodiment, the invention provides a method, use,
device or formulation of any of the preceding claims, wherein the
disease or disorder associated with angiogenesis is selected from:
tumor or cancer growth (neoplasia), skin disorders,
neovascularization, and inflammatory and arthritic diseases. In a
certain embodiment, the disease or disorder associated with
angiogenesis is tumor or cancer growth (neoplasia). In a certain
embodiment, the disease or disorder is: eye or ocular cancer,
rectal cancer, colon cancer, cervical cancer, prostate cancer,
breast cancer and bladder cancer, oral cancer, benign and malignant
tumors, stomach cancer, liver cancer, pancreatic cancer, lung
cancer, corpus uteri, ovary cancer, prostate cancer, testicular
cancer, renal cancer, brain/cns cancer (e.g., gliomas), throat
cancer, skin melanoma, acute lymphocytic leukemia, acute
myelogenous leukemia, Ewing's Sarcoma, Kaposi's Sarcoma, basal cell
carinoma and squamous cell carcinoma, small cell lung cancer,
choriocarcinoma, rhabdomyosarcoma, angiosarcoma,
hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx
cancer, esophageal cancer, larynx cancer, lymphoma,
neurofibromatosis, tuberous sclerosis, hemangiomas, and
lymphangiogenesis.
In a certain embodiment, the disease or disorder associated with
angiogenesis is a skin disorder. In a certain embodiment, the
disease or disorder is: psoriasis, acne, rosacea, warts, eczema,
hemangiomas, lymphangiogenesis, Sturge-Weber syndrome, venous
ulcers of the skin, neurofibromatosis, and tuberous sclerosis.
In a certain embodiment, the disease or disorder associated with
angiogenesis is neovascularization. In certain embodiments, the
disease or disorder is: diabetic retinopathy, retinopathy of
prematurity, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasias, epidemic keratoconjunctivitis, vitamin A
deficiency, contact lens overwear, atopic keratitis, superior
limbic keratitis, pterygium keratitis sicca, Sjogren's, acne
rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid
degeneration, chemical burns, bacterial ulcers, fungal ulcers,
herpes simplex infections, herpes zoster infections, protozoan
infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal
degeneration, marginal keratolysis, trauma, rheumatoid arthritis,
systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,
Stevens-Johnson disease, pemphigoid, radial keratotomy, corneal
graft rejection, macular edema, macular degeneration, sickle cell
anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's
disease, vein occlusion, artery occlusion, carotid obstructive
disease, chronic uveitis/vitritis, mycobacterial infections, Lyme
disease, systemic lupus erythematosus, retinopathy of prematurity,
Eales' disease, Behcet's disease, infections causing a retinitis or
choroiditis, presumed ocular histoplasmosis, Best's disease,
myopia, optic pits, Stargardt's disease, pars planitis, chronic
retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma
and post-laser complications, and diseases associated with rubeosis
(neovascularization of the ankle). In a certain embodiment, the
disease or disorder associated with angiogenesis is rheumatoid
arthritis, diabetic retinopathy, macular edema, or macular
degeneration.
In a certain embodiment, the disease or disorder associated with
angiogenesis is inflammatory and arthritic disease. In a certain
embodiment, the disease or disorder is: rheumatoid arthritis,
osteoarthritis, lupus, scleroderma, Crohn's disease, ulcerative
colitis, psoriasis, sarcoidosis, Sarcoidosis, skin lesions,
hemangiomas, Osler-Weber-Rendu disease, hereditary hemorrhagic
telangiectasia, and osteoarthritis.
In certain embodiments, the subject anti-angiogenic compounds are
used as part of a treatment or prevention for an optic neuropathy.
The compounds can be administered, for example, by for intraocular
injection or implantation. The anti-angiogenic compound can be
administered alone, or in combination with other agents, including
anti-inflammatory compounds, neuroprotective agents, agents that
reduce introcular pressure (TOP), and/or immunomodulatory
compounds. For instance, the anti-angiogenic compound can be
administered as part of therapy that includes treatment with a
cholinergic agonists, cholinesterase inhibitors, carbonic anhydrase
inhibitors, adrenergic agonists (such as alpha2-selective
adrenergic agonists), beta-blockers, prostaglandin analogues,
osmotic diuretics, p38 kinase antagonists, Cox-2 inhibitors,
corticosteroid (such as triamcinolone, dexamethasone, fluocinolone,
cortisone, prednisolone, flumetholone, or derivatives thereof such
as triamcinolone acetonide or fluocinolone acetonide), salts
thereof, isomers thereof, prodrugs thereof, and mixtures of any of
these.
As used herein, the terms "optic neuropathy", or "optic
neuropathies" are intended to include diseases, disorders, or
damage to the nerves or other structures of the eye. By way of
example, such optic neuropathies include uveitis, such as anterior
uveitis, intermediate uveitis, posterior uveitis, and diffuse
uveitis; uveitic syndromes, such as ankylosing spondylitis,
juvenile rheumatoid arthritis, pars planitis, toxoplasmosis,
cytomegalovirus, inflammation caused by herpes zoster, inflammation
caused by herpes simplex, toxocariasis, birdshot chorioretinopathy,
presumed ocular histoplasmosis syndrome, syphilis, tuberculosis,
Vogt-Koyanagi-Harada syndrome, sympathetic ophthalmia, ocular
sarcoidosis and endophthalmitis; masquerade syndromes, such as
intraocular malignancy, retinitis pigmentosa, and reactions to
drugs; vascular retinopathies, such as hypertensive retinopathy,
diabetic retinopathy, central retinal artery occlusion, and central
retinal vein occlusion; age-related macular degeneration; retinitis
pigmentosa; glaucoma; ocular hypertension; optic nerve and pathway
disorders, such as papilledema, papillitis, retrobulbar neuritis,
toxic amblyopia, optic atrophy, bitemporal hemianopia, and
homonymous hemianopia. In certain preferred embodiments, the
subject anti-angiogenic compounds are used as part of a treatment
for uveitis, Diabetic Macular Edema (DME), Wet ARMD, and CMV
retinitis.
There are various sustained release drug delivery devices for
implantation in the eye and treating various eye diseases that can
be readily adapted for delivery of the subject anti-angiogenic
compounds. Examples are found in the following patents, the
disclosures of which are incorporated herein by reference: U.S.
2005/0137583 (Renner); U.S. 2004/0219181 (Viscasillas); U.S.
2004/0265356 (Mosack); U.S. 2005/0031669 (Shafiee); U.S.
2005/0137538 (Kunzler); U.S. 2002/0086051A1 (Viscasillas); U.S.
2002/0106395A1 (Brubaker); U.S. 2002/0110591A1 (Brubaker et al.);
U.S. 2002/0110592A1 (Brubaker et al.); U.S. 2002/0110635A1
(Brubaker et al.); U.S. Pat. No. 5,378,475 (Smith et al.); U.S.
Pat. No. 5,773,019 (Ashton et al.); U.S. Pat. No. 5,902,598 (Chen
et al.); U.S. Pat. No. 6,001,386 (Ashton et al.); U.S. Pat. No.
6,726,918 (Wong); U.S. Pat. No. 6,331,313 (Wong); U.S. Pat. No.
5,824,072 (Wong); U.S. Pat. No. 5,632,984 (Wong); U.S. Pat. No.
6,217,895 (Guo et al.); U.S. Pat. No. 6,375,972 (Guo et al.). In
certain embodiments, the device include an inner drug core
including the anti-angiogenic compound, and some type of holder for
the drug core made of an impermeable material such as silicone or
other hydrophobic materials. The holder includes one or more
openings for passage of the pharmaceutically agent through the
impermeable material to eye tissue. Many of these devices include
at least one layer of material permeable to the active agent, such
as polyvinyl alcohol.
In still another embodiment, the invention provides a process for
preparing of 4R-cis-itraconazole or 45-cis-itraconazole comprising
the step of:
reacting the a compound of the formula:
##STR00003##
wherein R is a protecting group
with a compound of the formula:
##STR00004##
in the presence of base. In this process, the compound of the
formula:
##STR00005##
may be prepared by reacting a compound of the formula
with a compound of the formula
##STR00006##
The individual stereoisomers of formulas
##STR00007##
may be obtained by separation techniques known in the art
including, but not limited to, chromatography, HPLC,
crystallization, recrystallization, double-recrystallization, and
so on.
In other embodiments, processes for preparing compounds of
structural Formulas A-H are provided. Compounds of structural
Formulas A
(4-(4-(4-(4-(((2S,4R)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((S)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one) or B
(4-(4-(4-(4-(((2S,4R)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((R)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one), may be prepared by:
reacting a compound of structural Formula I with (S)- or
(R)-1-(sec-butyl)-4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-1H-1,2,-
4-triazol-5(4H)-one, respectively in the presence of a base:
##STR00008##
Compounds of structural Formula C
(4-(4-(4-(4-(((2R,4S)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((S)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one) or D
(4-(4-(4-(4-(((2R,4S)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((R)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one), may be prepared by:
reacting a compound of structural Formula II with (S)- or
(R)-1-(sec-butyl)-4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-1H-1,2,-
4-triazol-5(4H)-one, respectively in the presence of a base:
##STR00009##
Compounds of structural Formula E
(4-(4-(4-(4-(((2S,4S)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((S)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one) or F
(4-(4-(4-(4-(((2S,4S)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((R)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one), may be prepared by:
reacting a compound of structural Formula III with (S)- or
(R)-1-(sec-butyl)-4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-1H-1,2,-
4-triazol-5(4H)-one, respectively in the presence of a base:
##STR00010##
Compounds of structural formula G
(4-(4-(4-(4-(((2R,4R)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((S)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one) or H
(4-(4-(4-(4-(((2R,4R)-2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichloroph-
enyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-((R)-sec-b-
utyl)-1H-1,2,4-triazol-5(4H)-one), may be prepared by:
reacting a compound of structural Formula IV with (S)- or
(R)-1-(sec-butyl)-4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-1H-1,2,-
4-triazol-5(4H)-one, respectively in the presence of a base:
##STR00011##
In certain aspects, R is a suitable leaving group, such as a
tosylate ester, sulfate ester, nitrate ester, phosphate ester,
carboxylate ester, and the like.
Similarly, the compound of the formula:
##STR00012##
may be prepared by reacting 1,2,4 triazole with a compound of the
formula in the
##STR00013## presence of base.
Also, the compound of the formula:
##STR00014##
may be prepared by a process, comprising:
a.) reacting a compound for the formula:
##STR00015##
wherein R.sup.1 is an alkyl protecting group
with para-chloro-nitrobenzene to form a compound of the
formula:
##STR00016##
b.) hydrogenating the product of a.);
c.) reacting the hydrogenation product of b.) with
phenylchloroformate to form a compound of the formula:
##STR00017##
d.) reacting the product of c.) with N'-sec-butylformohydrazine to
form a compound of the formula:
##STR00018## and
e.) deprotecting the product of d.).
Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the inhibition endothelial cell proliferation and
blocks in vivo angiogenesis by itraconazole. (a) Screening results
for 2,604 existing drugs on HUVEC proliferation at 10 .mu.M. (b)
Inhibition of HUVEC (.circle-solid.) and human foreskin fibroblast
(.largecircle.) proliferation by itraconazole. (c) Cell cycle
analysis of HUVEC showing G1/S arrest upon 4S-cis itraconazole
treatment.
FIG. 2 shows the effect of cholesterol on the inhibition of BAEC
proliferation by itraconazole and azalanstat. BAEC were incubated
in DMEM, 10% lipoprotein deficient serum (LPDS) with indicated
concentrations of itraconazole (a) azalanstat (b) or TNP-470 (c)
either alone or in combination with 40 .mu.g/ml free cholesterol
for 36 h. Cells were then pulsed with 1 .mu.Ci [.sup.3H]-thymidine
for 8 h before they are harvested for scintillation counting.
FIG. 3 shows the knockdown of 14DM in HUVEC inhibits proliferation.
(a) Western blot of knockdown of transiently-expressed 14DM
protein. (b) RT-PCR of 14DM knockdown HUVEC. c) Knockdown of human
14DM in HUVEC inhibits cell proliferation.
FIG. 4 shows the treatment of mice with itraconazole, 37.5
mg/kg/day, i.p. significantly inhibited angiogenesis as shown in
representative Matrigel plugs (a) and in 100.times. sections of
plugs harvested from mice (b). (c) Erythrocyte-filled blood vessels
were counted per 100.times. field (*p=0.01, n=6 vehicle, n=8
itraconazole).
FIG. 5 is a table that provides the chiral HPLC analysis data
(retention time) and optical rotation measurements of the eight
itraconazole stereoisomers.
FIG. 6 is a table that provides the high-resolution mass
spectrometry data for the eight itraconazole stereoisomers.
FIG. 7 is a table showing the potency of each of the eight isolated
itraconazole stereoisomers in snit-fungal and HUVEC biological
assays.
FIG. 8 shows overlayed HPLC chromatograms for the cis-dioxolane
series of itraconazole. The order of the chromatograms for the
stereoisomers from bottom to top is: 23a; 23d; 23b; and 23c.
FIG. 9 shows overlayed HPLC chromatograms for the trans-dioxolane
series of itraconazole. The order of the chromatograms for the
stereoisomers from bottom to top is: 23e; 23h; 23f; and 23g.
DETAILED DESCRIPTION OF THE INVENTION
Itraconazole, used clinically as an antifungal agent, potently
inhibits in vitro proliferation of human umbilical vein endothelial
cells (HUVEC) and angiogenesis in vivo. The target responsible for
itraconazole's antifungal activity is lanosterol
14.alpha.-demethylase (14DM), a key enzyme involved in the
biosynthesis of ergosterol, which is required for the integrity of
the fungal cell membrane. However, the role of human 14DM in the
inhibition of angiogenesis by itraconazole remains unclear. The
poor correlation between human 14DM inhibition and antiangiogenic
activity for several structurally related potent azole antifungal
drugs implies that other primary molecular target(s) might be
responsible for the antiangiogenic activity of itraconazole with
14DM making only a partial contribution.
Itraconazole contains three stereocenters, which can yield a total
of eight stereoisomers. The dioxolane ring harbors two chiral
centers, while the third one marked as 2' resides on the sec-butyl
side chain appended to the triazolone ring (Scheme 2).
As an anti-fungal drug, the pill and i.v. formulations of
itraconazole are supplied as a 1:1:1:1 mixture of four
cis-stereoisomers. Although the antifungal activity and metabolism
of individual cis stereoisomers of itraconazole have been reported,
the activity of the trans-stereoisomers, 23e-23h, have not been
disclosed to date. Furthermore, itraconazole has not been examined
for antiangiogenic activity in any of its stereochemically pure
forms. Previous efforts have been limited to the synthesis and
determination of the antiangiogenic activity of the epimeric
mixtures of 4R- and 4S-cis itraconazole. Thus, the complete role of
stereochemistry in the two activities of itraconazole has not been
addressed.
The present disclosure is based in part on the discovery that
isolated stereoisomers are significantly more effective in the
inhibition of growth of endothelial cells. In addition, this
increase in therapeutic effectiveness may allow for the use of
significantly lower doses, depending on the patient, the condition
or infection treated and the route of administration. The
anti-fungal activity of the trans-stereoisomers and the
antiangiogenic activity of the eight stereochemically pure forms of
itraconazole are described herein. Furthermore, the role of human
14.alpha.-demethylase (14DM) in the inhibition of angiogenesis by
itraconazole is provided.
To systematically explore the effect of absolute stereochemistry at
every chiral center of itraconazole on both antifungal and
antiangiogenic activity, all the eight stereoisomers were
synthesized and a comparison of their respective antiangiogenic and
antifungal activities was made. As disclosed herein, all eight
stereoisomers of itraconazole (23a-23h) have been synthesized and
evaluated for activity against human endothelial cell proliferation
and for antifungal activity against five fungal strains.
The total synthesis of 4S-cis-itraconazole and 4R-cis-itraconazole
is shown in Scheme 1.
##STR00019##
In Scheme 1, a diastereoselective ketalization of intermediate
compound 2 using a chirally pure glycerol monotosylate afforded the
chirally pure intermediates 3a and 3b.
Similarly, another intermediate, 10, was synthesized via a
five-step sequence starting from the piperazine precursor 4 and
4-chloronitrobenzene 5. The final coupling of tosylate 3a or 3b and
phenol 10 was carried out under basic conditions to give pure
4S-cis-itraconazole or 4R-cis-itraconazole in good yield.
The inhibitory activity of these diasteromers was then determined
using a HUVEC proliferation assay. The 4R-cis diastereomer
(IC.sub.50=0.056.+-.0.01 .mu.M) was found to be about 20-fold more
potent than the 4S-cis stereoisomer (IC.sub.50=1.1.+-.0.13 .mu.M).
In comparison, the racemic itraconazole has an IC.sub.50 of 0.16
.mu.M.
To systematically explore the effect of absolute stereochemistry at
every chiral center of itraconazole on both antifungal and
antiangiogenic activity, all eight stereoisomers were synthesized
and their antiangiogenic and antifungal activities compared.
##STR00020##
The total synthesis began by reduction of the nitro group in
N-(4-methoxyphenyl)-N-(4-nitrophenyl)-piperazine 6 (Scheme 2) using
a palladium-catalyzed transfer hydrogenation. The use of hydrazine
as a hydrogen source produced much higher yields of aniline 7
compared with ammonium formate. The amino group in 7 was
subsequently transformed into the triazolone via the
phenylcarbamate and semicarbazide intermediates. The
stereochemistry at 2' position in 23a-23h was inherited from the
optically pure starting material, (R)-(-)-2-butanol (17a) or
(S)-(+)-2-butanol (17b). In order to achieve a stereospecific
N-alkylation of triazolone 12 by tosylate displacement of 18a or
18b, the proton abstraction of triazolone nitrogen was conducted
using potassium carbonate in conjunction with 18-crown-6 in order
to enhance the nucleophilicity of the nitrogen anion by forming
loose ion-pairs. Construction of the 1,3-dioxolane ring in 22a-22d
was achieved by acid-assisted ketalization of
2,2',4'-trichloroacetophenone 2 with optically pure glyceryl
tosylate 21a or 21b. While the stereochemistry at C-4 in 22a-22d
emanates from the chiral starting material 21a or 21b, C-2, the new
chiral center, is generated during ketalization. The ratio of cis-
versus trans-diastereomers 22a/22c or 22b/22d is dictated by the
steric effects, and a preponderance of cis-dioxolane is typically
afforded. The cis-diastereomer (22a or 22b) was separated from the
trans-diastereomer (22c or 22d) and further purified by
double-recrystallization.
The significant influence of stereochemistry at one end of
itraconazole on its activity suggests that this part of
itraconazole may participate in a stereospecific interaction with
target(s) in endothelial cells. Thus in some aspects, the invention
provides the use of chirally pure 4S-cis-itraconazole.
Nevertheless, in other embodiments, the invention provides the use
of chirally pure 4R-cis-itraconazole. As used herein, the term
"chirally pure" means substantially free of any other stereoisomer.
For a compound to be substantially free of any other stereoisomer
means that the compound is made up of a significantly greater
proportion of the indicated stereoisomer than of its optical
antipode (in the case of optical isomers), or any other
diastereomer (resulting when a compound has more than one
stereocenter). In some aspects of the invention, for a compound to
be substantially free of any other stereoisomer means that the
compound is made up of at least about 90% by weight of the
indicated stereoisomer and about 10% by weight or less of any other
stereoisomer. In still other aspects of the invention, for a
compound to be substantially free of any other stereoisomer means
that the compound is made up of at least about 95% by weight of the
indicated stereoisomer and about 5% by weight or less of any other
stereoisomer. In yet other aspects of the invention, for a compound
to be substantially free of any other stereoisomer means that the
compound is made up of at least about 99% by weight of the
indicated stereoisomer and about 1% by weight or less of any other
stereoisomer. In another aspect of the invention, for a compound to
be substantially free of any other stereoisomer means that the
compound is made up of nearly 100% by weight of the indicated
stereoisomer. The above percentages are based on the total amount
of the combined stereoisomers of the compound.
The present invention is based on the discovery that various
classes of compounds that have already been demonstrated as
tolerable in human patients as part of other therapies, also have
potent anti-angiogenic activities. In general, the compounds of the
present invention inhibit endothelial cell proliferation. In
certain preferred embodiments, the anti-angiogenic activity derives
at least in part from the ability of the compound to inhibit
progression through the G1/S point of the cell cycle.
In one aspect, the invention provides a method of inhibiting or
otherwise reducing angiogenesis in a subject using a treatment
protocol that includes administering a compound that inhibits
lanosterol 14.alpha.-demethylase (14DM). As described in further
detail below, it has been discovered that inhibition of 14DM in
endothelial cells can prevent their proliferation, and makes 14DM
inhibitors useful as anti-angiogenic agents. 14DM, catalyzes an
essential step in the biosynthesis of ergosterol required for the
membrane integrity of fungal cells. The demethylation of lanosterol
is a common step between fungi and humans in sterol biosynthesis
prior to the divergence of the pathways leading to ergosterol in
fungi and cholesterol in humans, respectively. Although
itraconazole as well as other azole antifungal drugs preferably
inhibit the fungal 14DM over its human counterparts, they do
inhibit the human enzyme at higher concentrations. The IC.sub.50
values of itraconazole for human 14DM varies from 0.61 .mu.M to 30
.mu.M for unknown reasons. While not wishing to be bound by any
particular theory, the inhibition of endothelial cell cycle by
itraconazole may be mediated at least in part through the
inhibition of human 14DM.
In the case of dioxolane-containing azole antifungals like
itraconazole, ketoconazole, and terconazole, it has been noted that
the cis-diastereomeric pairs exhibit much higher antifungal potency
over their trans counterparts and thus for efficacy reasons they
have been used clinically as mixtures of cis-diastereomers. Docking
studies performed based on fluconazole-MtCYP51 (referred to as 14DM
for human enzyme) crystal structure have offered an explanation to
this effect. Homology-modeled CaCYP51 complexed with different
stereoisomers of ketoconazole have been analyzed and it was
observed that the cis-pairs (2S4R and 2R4S) and only one of the
trans pairs, namely 2S4S-ketoconazole, avidly bind to CaCYP51,
which is in good agreement with reported IC.sub.50 values of the
stereoisomers of ketoconazole against C. albicans (J. Med. Chem.
1992; 35: 2818-2825). Antifungal activities measured herein for the
eight stereoisomers of itraconazole against the three ascomycetes
perfectly match the pattern observed with ketoconazole. It is
possible that the CYP51 enzymes of ascomycetes poorly bind the 2R4R
itraconazole, whereas in the case of phylogenetically distant C.
neoformans, this scenario of binding among the trans-pairs is quite
the opposite. This may also be explained by the expression of a
stereoselective efflux pump or catabolic enzyme in this strain.
Taken together, these data indicate that unlike HUVEC inhibition,
the sensitivity of fungal growth to itraconazole is dictated not by
cis-trans configuration of the dioxolane ring but instead by the
absolute stereochemistry at the 2 and 4 carbons. The only
commonality that was observed for the role of stereochemistry in
HUVEC and fungal inhibition was that the stereochemistry at the 2'
position had little influence on potency in either case.
All the cis-diastereomers, which make up the commercial
itraconazole, exhibited high potency in both HUVEC and fungal
inhibition. All the trans diastereoisomers were less potent in
HUVEC proliferation than were the cis diastereoisomers. In
contrast, one pair of trans diastereoisomers, 23e and 23f, were
roughly as potent as the cis-diastereomers with respect to
antifungal activity against four out of five strains. The lack of
correlation between HUVEC and fungal sensitivity to optically pure
itraconazole stereoisomers suggests that human 14DM is not likely
to be the major target for the antiangiogenic activity of
itraconazole. There is evidence that suggests that the inhibitory
effect of itraconazole on endothelial cells results largely from
its inhibition of cholesterol trafficking through the lysosomal
compartment, leading to inhibition of the mTOR pathway. The results
disclosed herein provide previously unavailable data on the role of
stereochemistry in the potency of itraconazole against an emerging
therapeutic target for this drug, angiogenesis. Distinct
antiangiogenic and antifungal activity profiles of the
trans-stereoisomers, provided herein, especially 23e and 23f,
suggest different molecular mechanisms underlying the
anti-angiogenic and anti-fungal activities of itraconazole.
Compounds 23a and 23b possess the greatest antiangiogenic potential
and therefore are good candidates for lead compounds for further
optimization of itraconazole as an antiangiogenic drug.
Thus, in one aspect, the invention provides a method of inhibiting
angiogenesis in a subject, the method comprising the step of
administering to the subject an angiogenesis-inhibiting compound,
wherein the compound is an inhibitor of 14DM.
In yet another aspect, the invention provides a method of treating
a subject identified as suffering from or susceptible to a disease
or disorder associated with angiogenesis, the method comprising the
step of administering to the subject a therapeutic amount of an
angiogenesis-inhibiting compound, wherein the compound is an
inhibitor of 14DM.
In still another aspect, the invention provides a method of
inhibiting angiogenesis in a subject, the method comprising the
step of administering to the subject an angiogenesis-inhibiting
compound, wherein the compound is 4S-cis-itraconazole.
In yet another aspect, the invention provides a method of treating
a subject identified as suffering from or susceptible to a disease
or disorder associated with angiogenesis, the method comprising the
step of administering to the subject a therapeutic amount of an
angiogenesis-inhibiting compound, wherein the compound is
4S-cis-itraconazole.
In still another aspect, the invention provides a method of
inhibiting angiogenesis in a subject, the method comprising the
step of administering to the subject an angiogenesis-inhibiting
compound, wherein the compound is 4R-cis-itraconazole.
In yet another aspect, the invention provides a method of treating
a subject identified as suffering from or susceptible to a disease
or disorder associated with angiogenesis, the method comprising the
step of administering to the subject a therapeutic amount of an
angiogenesis-inhibiting compound, wherein the compound is
4R-cis-itraconazole.
In another aspect, the invention provides a method of inhibiting
angiogenesis in a subject, the method comprising the step of
administering to the subject an angiogenesis-inhibiting compound,
wherein the compound is azalanstat.
In yet another aspect, the invention provides a method of treating
a subject identified as suffering from or susceptible to a disease
or disorder associated with angiogenesis, the method comprising the
step of administering to the subject a therapeutic amount of an
angiogenesis-inhibiting compound, wherein the compound is
azalanstat.
In another aspect, the invention provides a method of inhibiting
angiogenesis in a subject, treating a subject identified as
suffering from or susceptible to a disease or disorder associated
with angiogenesis, or any method delineated herein, wherein the
subject is identified as to be in need of such treatment (e.g.,
angiogenesis reduction or inhibition).
In a certain embodiment, itraconazole inhibits angiogenesis by
inhibiting endothelial cell proliferation. In another embodiment,
itraconazole is a G1/S cell cycle inhibitor.
In a certain embodiment, the invention further comprises an
additional therapeutic agent. In a further embodiment, the
additional therapeutic agent is an angiogenesis-inhibiting
compound. In another further embodiment, the additional therapeutic
agent is an anticancer compound.
In a certain embodiment, the disease or disorder associated with
angiogenesis is selected from: tumor or cancer growth (neoplasia),
skin disorders, neovascularization, and inflammatory and arthritic
diseases.
In a certain embodiment, the step of administering the
angiogenesis-inhibiting compound comprises administering the
compound orally, topically, parentally, intravenously or
intramuscularly.
In a certain embodiment, the step of administering the
angiogenesis-inhibiting compound comprises administering the
compound in a dosage of between about 0.1 and 100 mg/kg/day. In
another embodiment, the step of administering the
angiogenesis-inhibiting compound comprises administering the
compound in a dosage of less than about 500 mg/day.
In a certain embodiments, the subject is an animal or human.
In a certain embodiment, the invention provides a kit comprising an
effective amount of an angiogenesis-inhibiting compound in unit
dosage form, together with instructions for administering the
angiogenesis-inhibiting compound to a subject suffering from or
susceptible to a disease or disorder or symptoms thereof associated
with angiogenesis. In a further embodiment, the
angiogenesis-inhibiting compound is MPA.
In a certain embodiment, the invention provides administering an
effective amount of a composition comprising an
angiogenesis-inhibiting compound and a pharmaceutically suitable
excipient.
This instant invention is based on the premise that there exist
unappreciated physiological activities among known clinical drugs
demonstrating inhibition of lanosterol 14.alpha.-demethylase. This
premise was proven by the identification of multiple known drugs
with unexpected inhibitory effects on endothelial cell
proliferation in vitro and angiogenesis in vivo. In addition to the
endothelial cell proliferation assay, a library in a number of
other cellular assays was screened. It was found that the hit rates
with this drug library are significantly higher than commercially
available small molecule libraries on more than half a dozen
cellular screens. The molecular basis of these high hit rates may
lie in the shared genome and largely overlapping proteome of all
human cell types and tissues. Significant redundancy exists in the
usage of individual genes in different physiological as well as
pathological processes. Thus, there is great potential in screening
existing drugs for novel biological and therapeutic activities.
The term "effective amount" is used throughout the specification to
describe concentrations or amounts of compounds according to the
present invention which may be used to produce a favorable change
in the disease or condition treated, whether that change is a
remission, a decrease in growth or size of cancer, tumor or other
growth, a favorable physiological result including the clearing up
of skin or tissue, or the like, depending upon the disease or
condition treated.
As used herein, the terms "prevent," "preventing," "prevention,"
and the like refer to reducing the probability of developing a
disorder or condition in a subject, who does not have, but is at
risk of or susceptible to developing a disorder or condition.
The term "subject" is used throughout the specification to describe
an animal, preferably a human, to whom treatment, including
prophylactic treatment, with the compounds according to the present
invention is provided. For treatment of those infections,
conditions or disease states which are specific for a specific
animal such as a human patient, the term patient refers to that
specific animal. In most instances, the term patient refers to a
human patient.
As used herein, the terms "treat," treating," "treatment," and the
like refer to reducing or ameliorating a disorder and/or symptoms
associated therewith. It will be appreciated that, although not
precluded, treating a disorder or condition does not require that
the disorder, condition or symptoms associated therewith be
completely eliminated.
As used herein, the terms "anti-angiogenic compound" and
"angiogenesis inhibiting compound" may be used interchangeably.
The term "tumor" is used to describe an abnormal growth in tissue
which occurs when cellular proliferation is more rapid than normal
tissue and continues to grow after the stimuli that initiated the
new growth cease. Tumors generally exhibit partial or complete lack
of structural organization and functional coordination with the
normal tissue, and usually form a distinct mass of tissue which may
be benign (benign tumor) or malignant (carcinoma). Tumors tend to
be highly vascularized.
The term "cancer" is used as a general term herein to describe
malignant tumors or carcinoma. These malignant tumors may invade
surrounding tissues, may metastasize to several sites and are
likely to recur after attempted removal and to cause death of the
patient unless adequately treated. As used herein, the terms
carcinoma and cancer are subsumed under the term tumor. Methods of
treating tumors and/or cancer according to the present invention
comprise administering to a patient in need thereof an effective
amount of one or compounds according to the present invention.
Angiogenesis is used throughout the specification to describe the
biological processes which result in the development of blood
vessels or increase in the vascularity of tissue in an organism.
Persistent, unregulated angiogenesis occurs in a multiplicity of
disease states, tumor metastasis and abnormal growth by endothelial
cells and supports the pathological damage seen in these
conditions. The diverse pathological states created due to
unregulated angiogenesis have been grouped together as angiogenic
dependent or angiogenic associated diseases. Therapies directed at
control of the angiogenic processes could lead to the abrogation or
mitigation of these diseases.
Diseases or disorders treated, ameliorated or prevented by the
instant invention include the following: neoplasia, internal
malignancies such as eye or ocular cancer, rectal cancer, colon
cancer, cervical cancer, prostate cancer, breast cancer and bladder
cancer, benign and malignant tumors, including various cancers such
as, anal and oral cancers, stomach, rectal, liver, pancreatic,
lung, cervix uteri, corpus uteri, ovary, prostate, testis, renal,
mouth/pharynx, esophageal, larynx, kidney, brain/cns (e.g.,
gliomas), head and neck, throat, skin melanoma, acute lymphocytic
leukemia, acute myelogenous leukemia, Ewing's Sarcoma, Kaposi's
Sarcoma, basal cell carinoma and squamous cell carcinoma, small
cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma,
hemangioendothelioma, Wilms Tumor, neuroblastoma, lymphoma,
neurofibromatosis, tuberous sclerosis (each of which conditions
produces benign tumors of the skin), hemangiomas,
lymphangiogenesis, rhabdomyosarcomas, retinoblastoma, osteosarcoma,
acoustic neuroma, neurofibroma, trachoma, pyogenic granulomas,
blood-born tumors such as leukemias, any of various acute or
chronic neoplastic diseases of the bone marrow in which
unrestrained proliferation of white blood cells occurs, usually
accompanied by anemia, impaired blood clotting, and enlargement of
the lymph nodes, liver, and spleen, psoriasis, acne, rosacea,
warts, eczema, neurofibromatosis, Sturge-Weber syndrome, venous
ulcers of the skin, tuberous sclerosis, chronic inflammatory
disease, arthritis, lupus, scleroderma, diabetic retinopathy,
retinopathy of prematurity, corneal graft rejection, neovascular
glaucoma and retrolental fibroplasias, epidemic
keratoconjunctivitis, vitamin A deficiency, contact lens overwear,
atopic keratitis, superior limbic keratitis, pterygium, keratitis
sicca, Sjogren's, phylectenulosis, syphilis, Mycobacteria
infections, lipid degeneration, chemical burns, bacterial ulcers,
fungal ulcers, herpes simplex infections, herpes zoster infections,
protozoan infections, Mooren's ulcer, Terrien's marginal
degeneration, marginal keratolysis, trauma, rheumatoid arthritis,
systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,
Stevens-Johnson disease, pemphigoid, radial keratotomy, corneal
graft rejection, diabetic retinopathy, macular edema, macular
degeneration, sickle cell anemia, sarcoid, pseudoxanthoma
elasticum, Paget's disease, vein occlusion, artery occlusion,
carotid obstructive disease, chronic uveitis/vitritis, Lyme
disease, systemic lupus erythematosus, Eales' disease, Behcet's
disease, infections causing a retinitis or choroiditis, presumed
ocular histoplasmosis, Best's disease, myopia, optic pits,
Stargardt's disease, pars planitis, chronic retinal detachment,
hyperviscosity syndromes, toxoplasmosis, trauma, post-laser
complications, rubeosis (neovascularization of the ankle), diseases
caused by the abnormal proliferation of fibrovascular or fibrous
tissue including all forms of proliferative vitreoretinopathy,
whether or not associated with diabetes, neovascular disease,
pannus, diabetic macular edema, vascular retinopathy, retinal
degeneration, inflammatory diseases of the retina, proliferative
vitreoretinopathy, diseases associated with rubeosis
(neovascularization of the ankle), diseases caused by the abnormal
proliferation of fibrovascular or fibrous tissue including all
forms of proliferative vitreoretinopathy, Crohn's disease and
ulcerative colitis, sarcoidosis, osteoarthritis, inflammatory bowel
diseases, skin lesions, Osler-Weber-Rendu disease, or hereditary
hemorrhagic telangiectasia, osteoarthritis, Sarcoidosis, skin
lesions, acquired immune deficiency syndrome, and small bowel
obstruction.
The inhibition of angiogenesis in treating or reversing the disease
state or condition is an important aspect of the present invention.
More particularly, the present invention relates to methods for
inhibiting the growth of neoplasia, including a malignant tumor or
cancer comprising exposing the neoplasia to an inhibitory or
therapeutically effective amount or concentration of at least one
of the disclosed compounds. This method may be used
therapeutically, in the treatment of neoplasia, including cancer or
in comparison tests such as assays for determining the activities
of related analogs as well as for determining the susceptibility of
a patient's cancer to one or more of the compounds according to the
present invention. Treatment of internal malignancies such as eye
or ocular cancer, rectal cancer, colon cancer, cervical cancer,
prostate cancer, breast cancer and bladder cancer, among numerous
others, and oral malignancies are also contemplated by the present
invention.
Angiogenesis inhibiting compounds of the present invention are used
to treat, ameliorate or prevent benign and malignant tumors,
including various cancers such as, cervical, anal and oral cancers,
stomach, colon, bladder, rectal, liver, pancreatic, lung, breast,
cervix uteri, corpus uteri, ovary, prostate, testis, renal,
brain/cns (e.g., gliomas), head and neck, eye or ocular, throat,
skin melanoma, acute lymphocytic leukemia, acute myelogenous
leukemia, Ewing's Sarcoma, Kaposi's Sarcoma, basal cell carinoma
and squamous cell carcinoma, small cell lung cancer,
choriocarcinoma, rhabdomyosarcoma, angiosarcoma,
hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx,
esophageal, larynx, kidney and lymphoma, among others. In addition,
conditions such as neurofibromatosis, tuberous sclerosis (each of
which conditions produces benign tumors of the skin), hemangiomas
and lymphangiogenesis, among others, may be treated effectively
with compounds according to the present invention.
Angiogenesis is prominent in solid tumor formation and metastasis.
Angiogeneic factors have been found associated with several solid
tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,
neuroblastoma, and osteosarcoma. A tumor cannot expand without a
blood supply to provide nutrients and remove cellular wastes.
Tumors in which angiogenesis is important include solid tumors, and
benign tumors such as acoustic neuroma, neurofibroma, trachoma and
pyogenic granulomas. Prevention of angiogenesis could halt the
growth of these tumors and the resultant damage to the animal due
to the presence of the tumor.
It should be noted that angiogenesis has been associated with
blood-born tumors such as leukemias, any of various acute or
chronic neoplastic diseases of the bone marrow in which
unrestrained proliferation of white blood cells occurs, usually
accompanied by anemia, impaired blood clotting, and enlargement of
the lymph nodes, liver, and spleen. It is believed that
angiogenesis plays a role in the abnormalities in the bone marrow
that give rise to leukemia-like tumors.
Angiogenesis is important in two stages of tumor metastasis. The
first stage where angiogenesis stimulation is important is in the
vascularization of the tumor, which allows tumor cells to enter the
blood stream and to circulate throughout the body. After the tumor
cells have left the primary site, and have settled into the
secondary, metastasis site, angiogenesis must occur before the new
tumor can grow and expand. Therefore, prevention or control of
angiogenesis could lead to the prevention of metastasis of tumors
and possibly contain the neoplastic growth at the primary site.
Knowledge of the role of angiogenesis in the maintenance and
metastasis of tumors has led to a prognostic indicator for breast
cancer. The amount of neovascularization found in the primary tumor
was determined by counting the microvessel density in the area of
the most intense neovascularization in invasive breast carcinoma. A
high level of microvessel density was found to correlate with tumor
recurrence. Control of angiogenesis by therapeutic means can lead
to cessation of the recurrence of the tumors.
One of the most frequent angiogenic diseases of childhood is the
hemangioma. In most cases, the tumors are benign and regress
without intervention. In more severe cases, the tumors progress to
large cavernous and infiltrative forms and create clinical
complications. Systemic forms of hemangiomas, the hemangiomatoses,
have a high mortality rate. Therapy-resistant hemangiomas exist
that cannot be treated with therapeutics currently in use.
Angiogenic disease, angiogenic disorder and angiogenic skin
disorder are used throughout the specification to describe a
disorder, generally a skin disorder or related disorder which
occurs as a consequence of or which results in increased
vascularization in tissue. Any skin disorder which has as a primary
or secondary characterization, increased vascularization, is
considered an angiogenic skin disorder for purposes of the present
invention and is amenable to treatment with compounds according to
the present invention.
Methods for treating, ameliorating, or preventing angiogenic skin
disorders such as psoriasis, acne, rosacea, warts, eczema,
hemangiomas, lymphangiogenesis, neurofibromatosis, Sturge-Weber
syndrome, venous ulcers of the skin, tuberous sclerosis, chronic
inflammatory disease and arthritis, as well as inflammation such as
chronic inflammatory disease, including arthritis, lupus and
scleroderma are also contemplated by the present invention, such
methods comprising administering a therapeutically effective amount
of one or more of the disclosed compounds to a patient in need of
such treatment.
Diseases associated with neovascularization include optic disc
neovascularization, iris neovascularization, retinal
neovascularization, choroidal neovascularization, corneal
neovascularization, and intravitreal neovascularization.
Diseases associated with corneal neovascularization and
retinal/choroidal neovascularization that can be treated,
ameliorated, or prevented, according to the present invention
include but are not limited to, diabetic retinopathy, retinopathy
of prematurity, corneal graft rejection, neovascular glaucoma and
retrolental fibroplasias, epidemic keratoconjunctivitis, vitamin A
deficiency, contact lens overwear, atopic keratitis, superior
limbic keratitis, pterygium keratitis sicca, Sjogren's, acne
rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid
degeneration, chemical burns, bacterial ulcers, fungal ulcers,
herpes simplex infections, herpes zoster infections, protozoan
infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal
degeneration, marginal keratolysis, trauma, rheumatoid arthritis,
systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,
Stevens-Johnson disease, pemphigoid, radial keratotomy, macular
edema, macular degeneration, sickle cell anemia, sarcoid, syphilis,
pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery
occlusion, carotid obstructive disease, chronic uveitis/vitritis,
mycobacterial infections, Lyme disease, systemic lupus
erythematosus, retinopathy of prematurity, Eales' disease, Behcet's
disease, infections causing a retinitis or choroiditis, presumed
ocular histoplasmosis, Best's disease, myopia, optic pits,
Stargardt's disease, pars planitis, chronic retinal detachment,
hyperviscosity syndromes, toxoplasmosis, trauma and post-laser
complications. Other diseases include, but are not limited to,
diseases associated with rubeosis (neovascularization of the
ankle), diseases caused by the abnormal proliferation of
fibrovascular or fibrous tissue including all forms of
proliferative vitreoretinopathy, whether or not associated with
diabetes, and corneal graft rejection.
In some embodiments, the corneal neovascularization to be treated
or inhibited is caused by trauma, chemical burns or corneal
transplantation. In other particular embodiments, the iris
neovascularization to be treated or inhibited is caused by diabetic
retinopathy, vein occlusion, ocular tumor or retinal detachment. In
still other particular embodiments, the retinal or intravitreal
neovascularization to be treated or inhibited is caused by diabetic
retinopathy, vein occlusion, sickle cell retinopathy, retinopathy
of prematurity, retinal detachment, ocular ischemia or trauma.
Additional diseases associated with choroidal neovascularization to
be treated or inhibited are caused by retinal or subretinal
disorders of age-related macular degeneration, diabetic macular
edema, presumed ocular histoplasmosis syndrome, myopic
degeneration, angioid streaks or ocular trauma.
One example of a disease mediated by angiogenesis is ocular
neovascular disease. This disease is characterized by invasion of
new blood vessels into the structures of the eye such as the retina
or cornea. It is the most common cause of blindness and is involved
in approximately twenty eye diseases. In age-related macular
degeneration, the associated visual problems are caused by an
ingrowth of chorioidal capillaries through defects in Bruch's
membrane with proliferation of fibrovascular tissue beneath the
retinal pigment epithelium.
Diseases associated with chronic inflammation and arthritis can be
treated, ameliorated or prevented by the compositions and methods
of the present invention. Diseases with symptoms of chronic
inflammation include inflammatory bowel diseases such as Crohn's
disease and ulcerative colitis, psoriasis, sarcoidosis, rheumatoid
arthritis, osteoarthritis, lupus and scleroderma. Angiogenesis is a
key element that these chronic inflammatory diseases have in
common. The chronic inflammation depends on continuous formation of
capillary sprouts to maintain an influx of inflammatory cells. The
influx and presence of the inflammatory cells produce granulomas
and thus, maintains the chronic inflammatory state.
The compositions and methods of the present invention can be used
to treat, ameliorate or prevent disease in patients with
inflammatory bowel diseases such as Crohn's disease and ulcerative
colitis. Both Crohn's disease and ulcerative colitis are
characterized by chronic inflammation and angiogenesis at various
sites in the gastrointestinal tract. Crohn's disease is
characterized by chronic granulomatous inflammation throughout the
gastrointestinal tract consisting of new capillary sprouts
surrounded by a cylinder of inflammatory cells. Prevention of
angiogenesis by the compositions and methods of the present
invention inhibits the formation of the sprouts and prevents the
formation of granulomas.
Chronic inflammation may also involve pathological angiogenesis.
Such disease states as ulcerative colitis and Crohn's disease show
histological changes with the ingrowth of new blood vessels into
the inflamed tissues. Bartonellosis, a bacterial infection found in
South America, can result in a chronic stage that is characterized
by proliferation of vascular endothelial cells. Another
pathological role associated with angiogenesis is found in
atherosclerosis. The plaques formed within the lumen of blood
vessels have been shown to have angiogenic stimulatory
activity.
Crohn's disease occurs as a chronic transmural inflammatory disease
that most commonly affects the distal ileum and colon but may also
occur in any part of the gastrointestinal tract from the mouth to
the anus and perianal area. Patients with Crohn's disease generally
have chronic diarrhea associated with abdominal pain, fever,
anorexia, weight loss and abdominal swelling. Ulcerative colitis is
also a chronic, nonspecific, inflammatory and ulcerative disease
arising in the colonic mucosa and is characterized by the presence
of bloody diarrhea.
The inflammatory bowel diseases also show extraintestinal
manifestations such as skin lesions. Such lesions are characterized
by inflammation and angiogenesis and can occur at many sites other
than the gastrointestinal tract. The compositions and methods of
the present invention are also capable of treating these lesions by
preventing the angiogenesis, thus reducing the influx of
inflammatory cells and the lesion formation.
Sarcoidosis is another chronic inflammatory disease that is
characterized as a multisystem granulomatous disorder. The
granulomas of this disease may form anywhere in the body and thus
the symptoms depend on the site of the granulomas and whether the
disease is active. The granulomas are created by the angiogenic
capillary sprouts providing a constant supply of inflammatory
cells.
The compositions and methods of the present invention can also
treat the chronic inflammatory conditions associated with
psoriasis. Psoriasis, a skin disease, is another chronic and
recurrent disease that is characterized by papules and plaques of
various sizes. Prevention of the formation of the new blood vessels
necessary to maintain the characteristic lesions leads to relief
from the symptoms.
Another disease (or symptoms thereof) which can be treated,
ameliorated or prevented according to the present invention is
rheumatoid arthritis. Rheumatoid arthritis is a chronic
inflammatory disease characterized by nonspecific inflammation of
the peripheral joints. It is believed that the blood vessels in the
synovial lining of the joints undergo angiogenesis. In addition to
forming new vascular networks, the endothelial cells release
factors and reactive oxygen species that lead to pannus growth and
cartilage destruction. The factors involved in angiogenesis may
actively contribute to, and help maintain, the chronically inflamed
state of rheumatoid arthritis. Another disease that can be treated
according to the present invention are Osler-Weber-Rendu disease,
or hereditary hemorrhagic telangiectasia, and acquired immune
deficiency syndrome.
Factors associated with angiogenesis may also have a role in
osteoarthritis. The activation of the chondrocytes by
angiogeneic-related factors contributes to the destruction of the
joint. At a later stage, the angiogeneic factors would promote new
bone formation. Therapeutic intervention that prevents the bone
destruction could halt the progress of the disease and provide
relief for persons suffering with arthritis.
Angiogenesis is also responsible for damage found in hereditary
diseases such as Osler-Weber-Rendu disease, or hereditary
hemorrhagic telangiectasia. This is an inherited disease
characterized by multiple small angiomas, tumors of blood or lymph
vessels. The angiomas are found in the skin and mucous membranes,
often accompanied by epistaxis (nosebleeds) or gastrointestinal
bleeding and sometimes with pulmonary or hepatic arteriovenous
fistula.
Angiogenesis is also involved in normal physiological processes
such as reproduction and wound healing. Angiogenesis is an
important step in ovulation and also in implantation of the
blastula after fertilization. Prevention of angiogenesis could be
used to induce amenorrhea, to block ovulation or to prevent
implantation by the blastula, thereby preventing conception.
In wound healing, excessive repair or fibroplasia can be a
detrimental side effect of surgical procedures and may be caused or
exacerbated by angiogenesis. Adhesions are a frequent complication
of surgery and lead to problems such as small bowel
obstruction.
The present compounds may be used to treat subjects including
animals, and in particular, mammals, including humans, as patients.
Thus, humans and other animals, and in particular, mammals,
suffering from diseases or disorders related to angiogenesis, can
be treated, ameliorated or prevented by administering to the
patient an effective amount of one or more of the compounds
according to the present invention or its derivative or a
pharmaceutically acceptable salt thereof optionally in a
pharmaceutically acceptable carrier or diluent, either alone, or in
combination with other known pharmaceutical agents (depending upon
the disease to be treated). Treatment according to the present
invention can also be administered in conjunction with other
conventional therapies, e.g., cancer therapy, such as radiation
treatment or surgery.
The present invention is also directed to pharmaceutical
compositions comprising an effective amount of one or more
compounds according to the present invention (including a
pharmaceutically acceptable salt, thereof), optionally in
combination with a pharmaceutically acceptable carrier, excipient
or additive.
A "pharmaceutically acceptable derivative or prodrug" means any
pharmaceutically acceptable salt, ester, salt of an ester, or other
derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. Particularly favored
derivatives and prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal (e.g., by allowing an orally
administered compound to be more readily absorbed into the blood)
or which enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system) relative to the
parent species.
While the angiogenesis inhibiting compounds of the invention can be
administered as the sole active pharmaceutical agent, they can also
be used in combination with one or more compounds of the invention
or other agents. When administered as a combination, the
therapeutic agents can be formulated as separate compositions that
are given at the same time or different times, or the therapeutic
agents can be given as a single composition.
The angiogenesis inhibiting compounds of the present invention may
be administered orally, parenterally, by inhalation spray,
rectally, vaginally, or topically in dosage unit formulations
containing conventional pharmaceutically acceptable carriers,
adjuvants, and vehicles. The term parenteral as used herein
includes, subcutaneous, intravenous, intramuscular, intrasternal,
infusion techniques, intraperitoneally, eye or ocular, intrabuccal,
transdermal, intranasal, into the brain, including intracranial and
intradural, into the joints, including ankles, knees, hips,
shoulders, elbows, wrists, directly into tumors, and the like, and
in suppository form.
The pharmaceutically active compounds of this invention can be
processed in accordance with conventional methods of pharmacy to
produce medicinal agents for administration to patients, including
humans and other mammals.
Modifications of the active compound can affect the solubility,
bioavailability and rate of metabolism of the active species, thus
providing control over the delivery of the active species. Further,
the modifications can affect the anti-angiogenesis activity of the
compound, in some cases increasing the activity over the parent
compound. This can easily be assessed by preparing the derivative
and testing its activity according to known methods well within the
routineer's skill in the art.
Pharmaceutical compositions based upon these chemical compounds
comprise the above-described compounds in a therapeutically
effective amount for treating diseases and conditions which have
been described herein, optionally in combination with a
pharmaceutically acceptable additive, carrier and/or excipient. One
of ordinary skill in the art will recognize that a therapeutically
effective amount of one of more compounds according to the present
invention will vary with the infection or condition to be treated,
its severity, the treatment regimen to be employed, the
pharmacokinetics of the agent used, as well as the patient (animal
or human) treated.
To prepare the pharmaceutical compositions according to the present
invention, a therapeutically effective amount of one or more of the
compounds according to the present invention is preferably
intimately admixed with a pharmaceutically acceptable carrier
according to conventional pharmaceutical compounding techniques to
produce a dose. A carrier may take a wide variety of forms
depending on the form of preparation desired for administration,
e.g., oral, topical or parenteral, including gels, creams
ointments, lotions and time released implantable preparations,
among numerous others. In preparing pharmaceutical compositions in
oral dosage form, any of the usual pharmaceutical media may be
used. Thus, for liquid oral preparations such as suspensions,
elixirs and solutions, suitable carriers and additives including
water, glycols, oils, alcohols, flavouring agents, preservatives,
colouring agents and the like may be used. For solid oral
preparations such as powders, tablets, capsules, and for solid
preparations such as suppositories, suitable carriers and additives
including starches, sugar carriers, such as dextrose, mannitol,
lactose and related carriers, diluents, granulating agents,
lubricants, binders, disintegrating agents and the like may be
used. If desired, the tablets or capsules may be enteric-coated or
sustained release by standard techniques.
The active compound is included in the pharmaceutically acceptable
carrier or diluent in an amount sufficient to deliver to a patient
a therapeutically effective amount for the desired indication,
without causing serious toxic effects in the patient treated.
Oral compositions will generally include an inert diluent or an
edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound or its prodrug derivative can
be incorporated with excipients and used in the form of tablets,
troches, or capsules. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition.
The tablets, pills, capsules, troches and the like can contain any
of the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a dispersing agent
such as alginic acid or corn starch; a lubricant such as magnesium
stearate; a glidant such as colloidal silicon dioxide; a sweetening
agent such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring. When the dosage
unit form is a capsule, it can contain, in addition to material-of
the above type, a liquid carrier such as a fatty oil. In addition,
dosage unit forms can contain various other materials which modify
the physical form of the dosage unit, for example, coatings of
sugar, shellac, or enteric agents.
Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil emulsion and as a
bolus, etc.
A tablet may be made by compression or molding, optionally with one
or more accessory ingredients. Compressed tablets may be prepared
by compressing in a suitable machine the active ingredient in a
free-flowing form such as a powder or granules, optionally mixed
with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets optionally may
be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein.
Methods of formulating such slow or controlled release compositions
of pharmaceutically active ingredients, are known in the art and
described in several issued US patents, some of which include, but
are not limited to, U.S. Pat. Nos. 3,870,790; 4,226,859; 4,369,172;
4,842,866 and 5,705,190, the disclosures of which are incorporated
herein by reference in their entireties. Coatings can be used for
delivery of compounds to the intestine (see, e.g., U.S. Pat. Nos.
6,638,534, 5,541,171, 5,217,720, and 6,569,457, and references
cited therein).
The active compound or pharmaceutically acceptable salt thereof may
also be administered as a component of an elixir, suspension,
syrup, wafer, chewing gum or the like. A syrup may contain, in
addition to the active compounds, sucrose or fructose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose.
In one embodiment, the active compounds are prepared with carriers
that will protect the compound against rapid elimination from the
body, such as a controlled release formulation, including implants
and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
A skilled artisan will recognize that in addition to tablets, other
dosage forms can be formulated to provide slow or controlled
release of the active ingredient. Such dosage forms include, but
are not limited to, capsules, granulations and gel-caps.
Liposomal suspensions may also be pharmaceutically acceptable
carriers. These may be prepared according to methods known to those
skilled in the art. For example, liposomal formulations may be
prepared by dissolving appropriate lipid(s) in an inorganic solvent
that is then evaporated, leaving behind a thin film of dried lipid
on the surface of the container. An aqueous solution of the active
compound are then introduced into the container. The container is
then swirled by hand to free lipid material from the sides of the
container and to disperse lipid aggregates, thereby forming the
liposomal suspension. Other methods of preparation well known by
those of ordinary skill may also be used in this aspect of the
present invention.
The formulations may conveniently be presented in unit dosage form
and may be prepared by conventional pharmaceutical techniques. Such
techniques include the step of bringing into association the active
ingredient and the pharmaceutical carrier(s) or excipient(s). In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
Formulations and compositions suitable for topical administration
in the mouth include lozenges comprising the ingredients in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
ingredient to be administered in a suitable liquid carrier.
Formulations suitable for topical administration to the skin may be
presented as ointments, creams, gels and pastes comprising the
ingredient to be administered in a pharmaceutical acceptable
carrier. A preferred topical delivery system is a transdermal patch
containing the ingredient to be administered.
Formulations for rectal administration may be presented as a
suppository with a suitable base comprising, for example, cocoa
butter or a salicylate.
Formulations suitable for nasal administration, wherein the carrier
is a solid, include a coarse powder having a particle size, for
example, in the range of 20 to 500 microns which is administered in
the manner in which snuff is administered, i.e., by rapid
inhalation through the nasal passage from a container of the powder
held close up to the nose. Suitable formulations, wherein the
carrier is a liquid, for administration, as for example, a nasal
spray or as nasal drops, include aqueous or oily solutions of the
active ingredient.
Formulations suitable for vaginal administration may be presented
as pessaries, tampons, creams, gels, pastes, foams or spray
formulations containing in addition to the active ingredient such
carriers as are known in the art to be appropriate.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic. If
administered intravenously, preferred carriers include, for
example, physiological saline or phosphate buffered saline
(PBS).
For parenteral formulations, the carrier will usually comprise
sterile water or aqueous sodium chloride solution, though other
ingredients including those which aid dispersion may be included.
Of course, where sterile water is to be used and maintained as
sterile, the compositions and carriers must also be sterilized.
Injectable suspensions may also be prepared, in which case
appropriate liquid carriers, suspending agents and the like may be
employed.
Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
antioxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, water for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
may be prepared from sterile powders, granules and tablets of the
kind previously described.
Administration of the active compound may range from continuous
(intravenous drip) to several oral administrations per day (for
example, Q.I.D.) and may include oral, topical, eye or ocular,
parenteral, intramuscular, intravenous, sub-cutaneous, transdermal
(which may include a penetration enhancement agent), buccal and
suppository administration, among other routes of administration,
including through an eye or ocular route.
Application of the subject therapeutics may be local, so as to be
administered at the site of interest. Various techniques can be
used for providing the subject compositions at the site of
interest, such as injection, use of catheters, trocars,
projectiles, pluronic gel, stents, sustained drug release polymers
or other device which provides for internal access. Where an organ
or tissue is accessible because of removal from the patient, such
organ or tissue may be bathed in a medium containing the subject
compositions, the subject compositions may be painted onto the
organ, or may be applied in any convenient way.
The angiogenesis-inhibiting compound may be administered through a
device suitable for the controlled and sustained release of a
composition effective in obtaining a desired local or systemic
physiological or pharmacological effect. The method includes
positioning the sustained released drug delivery system at an area
wherein release of the agent is desired and allowing the agent to
pass through the device to the desired area of treatment.
More specifically, the angiogenesis-inhibiting compound is
administered through an ocular device suitable for direct
implantation into the vitreous of the eye. Such devices of the
present invention are surprisingly found to provide sustained
controlled release of various compositions to treat the eye without
risk of detrimental local and systemic side effects. An object of
the present ocular method of delivery is to maximize the amount of
drug contained in an intraocular device while minimizing its size
in order to prolong the duration of the implant. See, e.g., U.S.
Pat. Nos. 5,378,475; 5,773,019; 6,001,386; 6,217,895, 6,375,972,
and 6,756,058 and U.S. Publications 20050096290 and
200501269448.
Other methods of delivery include: an ocular delivery system that
could be applied to an intra-ocular lens to prevent inflammation or
posterior capsular opacification, an ocular delivery system that
could be inserted directly into the vitreous, under the retina, or
onto the sclera, and wherein inserting can be achieved by injecting
the system or surgically implanting the system, a sustained release
drug delivery system, and a method for providing controlled and
sustained administration of an agent effective in obtaining a
desired local or systemic physiological or pharmacological effect
comprising surgically implanting a sustained release drug delivery
system at a desired location.
Examples include, but are not limited to the following: a sustained
release drug delivery system comprising an inner reservoir
comprising an effective amount of an agent effective in obtaining a
desired local or systemic physiological or pharmacological effect,
an inner tube impermeable to the passage of said agent, said inner
tube having first and second ends and covering at least a portion
of said inner reservoir, said inner tube sized and formed of a
material so that said inner tube is capable of supporting its own
weight, an impermeable member positioned at said inner tube first
end, said impermeable member preventing passage of said agent out
of said reservoir through said inner tube first end, and a
permeable member positioned at said inner tube second end, said
permeable member allowing diffusion of said agent out of said
reservoir through said inner tube second end; a method for
administering a compound of the invention to a segment of an eye,
the method comprising the step of implanting a sustained release
device to deliver the compound of the invention to the vitreous of
the eye or an implantable, sustained release device for
administering a compound of the invention to a segment of an eye; a
sustained release drug delivery device comprising: a) a drug core
comprising a therapeutically effective amount of at least one first
agent effective in obtaining a diagnostic effect or effective in
obtaining a desired local or systemic physiological or
pharmacological effect; b) at least one unitary cup essentially
impermeable to the passage of said agent that surrounds and defines
an internal compartment to accept said drug core, said unitary cup
comprising an open top end with at least one recessed groove around
at least some portion of said open top end of said unitary cup; c)
a permeable plug which is permeable to the passage of said agent,
said permeable plug is positioned at said open top end of said
unitary cup wherein said groove interacts with said permeable plug
holding it in position and closing said open top end, said
permeable plug allowing passage of said agent out of said drug
core, through said permeable plug, and out said open top end of
said unitary cup; and d) at least one second agent effective in
obtaining a diagnostic effect or effective in obtaining a desired
local or systemic physiological or pharmacological effect; or a
sustained release drug delivery device comprising: an inner core
comprising an effective amount of an agent having a desired
solubility and a polymer coating layer, the polymer layer being
permeable to the agent, wherein the polymer coating layer
completely covers the inner core.
The methods are particularly suitable for treating ocular
conditions such as glaucoma, proliferative vitreoretinopathy,
macular edema, including diabetic macular edema, age-related
macular degeneration, diabetic retinopathy, uveitis, ocular
neovascularization, and ocular infection. The devices are also
particularly suitable for use as an ocular device in treating
subjects suffering from ocular histoplasmosis, wherein the device
is surgically implanted within the vitreous of the eye.
The angiogenesis-inhibiting compound may be utilized in combination
with at least one known other therapeutic agent, or a
pharmaceutically acceptable salt of said agent. Examples of known
therapeutic agents which can be used for combination therapy
include, but are not limited to, corticosteroids (e.g., cortisone,
prodnisone, dexamethasone), non-steroidal anti-inflammatory drugs
(NSAIDS) (e.g., ibuprofen, celecoxib, aspirin, indomethicin,
naproxen), alkylating agents such as busulfan, cis-platin,
mitomycin C, and carboplatin; antimitotic agents such as
colchicine, vinblastine, paclitaxel, and docetaxel; topo I
inhibitors such as camptothecin and topotecan; topo II inhibitors
such as doxorubicin and etoposide; RNA/DNA antimetabolites such as
5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites
such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea and
thioguanine; antibodies such as Herceptin.RTM. and Rituxan.RTM..
Other known anti-cancer agents which can be used for combination
therapy include melphalan, chlorambucil, cyclophosamide,
ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin,
bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide,
retinoic acid, tamoxifen and alanosine.
The active compound or pharmaceutically acceptable salts thereof
can also be mixed with other active materials that do not impair
the desired action, or with materials that supplement the desired
action, such as other anticancer agents, and in certain instances
depending upon the desired therapy or target, antibiotics,
antifungals, antinflammatories, antiviral compounds or other agents
having a distinct pharmacological effect.
Alternatively, the compound of the invention may be administered
apart from the at least one known cancer chemotherapeutic agent. In
one embodiment, the compound of the invention and the at least one
known cancer chemotherapeutic agent are administered substantially
simultaneously, i.e., the compounds are administered at the same
time or one after the other, so long as the compounds reach
therapeutic levels in the blood at the same time. On another
embodiment, the compound of the invention and the at least one
known cancer chemotherapeutic agent are administered according to
their individual dose schedule, so long as the compounds reach
therapeutic levels in the blood.
It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of the present
invention may include other agents conventional in the art having
regard to the type of formulation in question, for example, those
suitable for oral administration may include flavoring agents.
In certain pharmaceutical dosage forms, the pro-drug form of the
compounds may be preferred. One of ordinary skill in the art will
recognize how to readily modify the present compounds to pro-drug
forms to facilitate delivery of active compounds to a targeted site
within the host organism or patient. The routineer also will take
advantage of favorable pharmacokinetic parameters of the pro-drug
forms, where applicable, in delivering the present compounds to a
targeted site within the host organism or patient to maximize the
intended effect of the compound.
Preferred prodrugs include derivatives where a group which enhances
aqueous solubility or active transport through the gut membrane is
appended to the structure of formulae described herein. See, e.g.,
Alexander, J. et al. Journal of Medicinal Chemistry 1988, 31,
318-322; Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam,
1985; pp 1-92; Bundgaard, H.; Nielsen, N. M. Journal of Medicinal
Chemistry 1987, 30, 451-454; Bundgaard, H. A Textbook of Drug
Design and Development; Harwood Academic Publ.: Switzerland, 1991;
pp 113-191; Digenis, G. A. et al. Handbook of Experimental
Pharmacology 1975, 28, 86-112; Friis, G. J.; Bundgaard, H. A
Textbook of Drug Design and Development; 2 ed.; Overseas Publ.:
Amsterdam, 1996; pp 351-385; Pitman, I. H. Medicinal Research
Reviews 1981, 1, 189-214. The prodrug forms may be active
themselves, or may be those such that when metabolized after
administration provide the active therapeutic agent in vivo.
Pharmaceutically acceptable salt forms may be the preferred
chemical form of compounds according to the present invention for
inclusion in pharmaceutical compositions according to the present
invention.
Certain of the compounds, in pharmaceutical dosage form, may be
used as agents for preventing a disease or condition from
manifesting itself. In certain pharmaceutical dosage forms, the
pro-drug form of the compounds according to the present invention
may be preferred. In particular, prodrug forms which rely on
C.sub.1 to C.sub.20 ester groups or amide groups (preferably a
hydroxyl, free amine or substituted nitrogen group) which may be
transformed into, for example, an amide or other group may be
particularly useful in this context.
The present compounds or their derivatives, including prodrug forms
of these agents, can be provided in the form of pharmaceutically
acceptable salts. As used herein, the term pharmaceutically
acceptable salts or complexes refers to appropriate salts or
complexes of the active compounds according to the present
invention which retain the desired biological activity of the
parent compound and exhibit limited toxicological effects to normal
cells. Nonlimiting examples of such salts are (a) acid addition
salts formed with inorganic acids (for example, hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and
the like), and salts formed with organic acids such as acetic acid,
oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic
acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and
polyglutamic acid, among others; (b) base addition salts formed
with metal cations such as zinc, calcium, sodium, potassium, and
the like, among numerous others.
The compounds herein are commercially available or can be
synthesized. As can be appreciated by the skilled artisan, further
methods of synthesizing the compounds of the formulae herein will
be evident to those of ordinary skill in the art. Additionally, the
various synthetic steps may be performed in an alternate sequence
or order to give the desired compounds. Synthetic chemistry
transformations and protecting group methodologies (protection and
deprotection) useful in synthesizing the compounds described herein
are known in the art and include, for example, those such as
described in R. Larock, Comprehensive Organic Transformations, 2nd.
Ed., Wiley-VCH Publishers (1999); T. W. Greene and P. G. M. Wuts,
Protective Groups in Organic Synthesis, 3rd. Ed., John Wiley and
Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents
for Organic Synthesis, John Wiley and Sons (1999); and L. Paquette,
ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and
Sons (1995), and subsequent editions thereof.
The compounds of this invention may contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of these compounds are expressly included
in the present invention. The compounds of this invention may also
be represented in multiple tautomeric forms, in such instances, the
invention expressly includes all tautomeric forms of the compounds
described herein (e.g., alkylation of a ring system may result in
alkylation at multiple sites, the invention expressly includes all
such reaction products). All such isomeric forms of such compounds
are expressly included in the present invention. All crystal forms
of the compounds described herein are expressly included in the
present invention.
Preferred unit dosage formulations are those containing a daily
dose or unit, daily sub-dose, as hereinabove recited, or an
appropriate fraction thereof, of the administered ingredient.
The dosage regimen for treating a disorder or a disease with the
angiogenesis inhibiting compounds of this invention and/or
compositions of this invention is based on a variety of factors,
including the type of disease, the age, weight, sex, medical
condition of the patient, the severity of the condition, the route
of administration, and the particular compound employed. Thus, the
dosage regimen may vary widely, but can be determined routinely
using standard methods.
The amounts and dosage regimens administered to a subject will
depend on a number of factors, such as the mode of administration,
the nature of the condition being treated, the body weight of the
subject being treated and the judgment of the prescribing
physician.
The amount of compound included within therapeutically active
formulations according to the present invention is an effective
amount for treating the infection or condition. In general, a
therapeutically effective amount of the present preferred compound
in dosage form usually ranges from slightly less than about 0.025
mg/kg/day to about 2.5 g/kg/day, preferably about 0.1 mg/kg/day to
about 100 mg/kg/day of the patient or considerably more, depending
upon the compound used, the condition or infection treated and the
route of administration, although exceptions to this dosage range
may be contemplated by the present invention. In its most preferred
form, compounds according to the present invention are administered
in amounts ranging from about 1 mg/kg/day to about 100 mg/kg/day.
In some aspects, a therapeutically effective amount of a compound
of the invention in a dosage form may be a therapeutically
effective low dose in the range of less than about 0.001 mg/kg/day
to about 10 mg/kg/day, preferably about 0.025 mg/kg/day to about 1
mg/kg/day of the patient or considerably less, depending upon the
compound used, the condition or infection treated and the route of
administration. The dosage of the compound will depend on the
condition being treated, the particular compound, and other
clinical factors such as weight and condition of the patient and
the route of administration of the compound. It is to be understood
that the present invention has application for both human and
veterinary use.
For oral administration to humans, a dosage of between
approximately 0.1 to 100 mg/kg/day, preferably between
approximately 1 and 100 mg/kg/day, is generally sufficient.
Where drug delivery is systemic rather than topical, this dosage
range generally produces effective blood level concentrations of
active compound ranging from less than about 0.04 to about 400
micrograms/cc or more of blood in the patient.
The compound is conveniently administered in any suitable unit
dosage form, including but not limited to one containing 1 to 3000
mg, preferably 5 to 500 mg of active ingredient per unit dosage
form. An oral dosage of 10-250 mg is usually convenient.
The concentration of active compound in the drug composition will
depend on absorption, distribution, inactivation, and excretion
rates of the drug as well as other factors known to those of skill
in the art. It is to be noted that dosage values will also vary
with the severity of the condition to be alleviated. It is to be
further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
composition. The active ingredient may be administered at once, or
may be divided into a number of smaller doses to be administered at
varying intervals of time.
In certain embodiments, the compound is administered once daily; in
other embodiments, the compound is administered twice daily; in yet
other embodiments, the compound is administered once every two
days, once every three days, once every four days, once every five
days, once every six days, once every seven days, once every two
weeks, once every three weeks, once every four weeks, once every
two months, once every six months, or once per year. The dosing
interval can be adjusted according to the needs of individual
patients. For longer intervals of administration, extended release
or depot formulations can be used.
The compounds of the invention can be used to treat diseases and
disease conditions that are acute, and may also be used for
treatment of chronic conditions. In certain embodiments, the
compounds of the invention are administered for time periods
exceeding two weeks, three weeks, one month, two months, three
months, four months, five months, six months, one year, two years,
three years, four years, or five years, ten years, or fifteen
years; or for example, any time period range in days, months or
years in which the low end of the range is any time period between
14 days and 15 years and the upper end of the range is between 15
days and 20 years (e.g., 4 weeks and 15 years, 6 months and 20
years). In some cases, it may be advantageous for the compounds of
the invention to be administered for the remainder of the patient's
life. In preferred embodiments, the patient is monitored to check
the progression of the disease or disorder, and the dose is
adjusted accordingly. In preferred embodiments, treatment according
to the invention is effective for at least two weeks, three weeks,
one month, two months, three months, four months, five months, six
months, one year, two years, three years, four years, or five
years, ten years, fifteen years, twenty years, or for the remainder
of the subject's life.
Still other objects, features, and attendant advantages of the
present invention will become apparent to those skilled in the art
from a reading of the preceding detailed description of embodiments
constructed in accordance therewith, taken in conjunction with the
accompanying drawings.
The invention also provides a pharmaceutical pack or kit comprising
one or more containers filled with one or more of the ingredients
of the pharmaceutical compositions of the invention. Associated
with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. In addition, the polypeptide of the present
invention may be employed on conjunction with other therapeutic
compounds.
The invention also provides kits for treatment or prevention of a
disease or disorder (or symptoms) thereof associated with
angiogenesis. In one embodiment, the kit includes an effective
amount of an angiogenesis-inhibiting compound in unit dosage form,
together with instructions for administering the
angiogenesis-inhibiting compound to a subject suffering from or
susceptible to a disease or disorder or symptoms thereof associated
with angiogenesis, wherein the effective amount of an
angiogenesis-inhibiting compound is less than 500 mg of the
compound. In preferred embodiments, the kit comprises a sterile
container which contains the angiogenesis-inhibiting compound; such
containers can be boxes, ampules, bottles, vials, tubes, bags,
pouches, blister-packs, or other suitable container form known in
the art. Such containers can be made of plastic, glass, laminated
paper, metal foil, or other materials suitable for holding
medicaments. The instructions will generally include information
about the use of the angiogenesis-inhibiting compound for treatment
of a disease or disorder or symptoms thereof associated with
angiogenesis; in preferred embodiments, the instructions include at
least one of the following: description of the
angiogenesis-inhibiting compound; dosage schedule and
administration for treatment of a disease or disorder or symptoms
thereof associated with angiogenesis; precautions; warnings;
indications; counter-indications; overdosage information; adverse
reactions; animal pharmacology; clinical studies; and/or
references. The instructions may be printed directly on the
container (when present), or as a label applied to the container,
or as a separate sheet, pamphlet, card, or folder supplied in or
with the container.
The following examples are intended to illustrate but not limit the
invention.
EXAMPLE 1
Synthesis of Cis-Itraconazole and Azalanstat
General Experimental
Thin-layer chromatography was performed on Merck precoated silica
gel 60F-254 plates and were visualized using 254 nm UV light, or by
staining with iodine, or eerie ammonium molybdate stain. Silica gel
(200-400 mesh, Merck) was used for flash chromatography. Reagents
were purchased from Aldrich, Acros, or Lancaster companies. Melting
points were recorded on a MeI-Temp II apparatus and are
uncorrected. NMR data were collected on a Varian Unity-400 (400 MHz
.sup.1H, 100 MHz .sup.13C) machine at the Department of
Pharmacology and Molecular Sciences, The Johns Hopkins University.
.sup.1H NMR spectra were obtained in deuteriochloroform
(CDCl.sub.3) with tetramethylsilane (TMS, .delta.=0.00 for .sup.1H)
or chloroform (.delta.=7.27 for .sup.1H), or
dimethylsulfoxide-d.sub.6 with tetramethylsilane (TMS, .delta.=0.00
for .sup.1H) as an internal reference. .sup.13C NMR spectra were
proton decoupled and were either in CDCl.sub.3 with either TMS
(.delta.=0.0 for .sup.13C) or chloroform (.delta.=77.0 for
.sup.13C), or dimethylsulfoxide-d.sub.6 with tetramethylsilane
(TMS, .delta.=0.00 for .sup.1H) as an internal reference. Chemical
shifts are reported in ppm (.delta.); multiplicities are indicated
by s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (doublet of doublet), dt (doublet of triplet), br.
(broad), app. (apparent) and exch. (exchangeable); coupling
constants, J, are reported in Hertz (Hz); integration is provided;
Data are presented in the form: chemical shift (multiplicity,
coupling constants, integration and assignments where relevant).
Low-resolution mass spectra were obtained on a Voyager DE-STR,
MALDI-TOF instrument at the AB Mass Spectrometry/Proteomics
Facility at the Johns Hopkins University. The MALDI-samples were
prepared by mixing droplets of the sample solutions in chloroform
or methanol and 2,5-dihydroxybenzoic acid solution in acetone,
where the latter served as the matrix. Data are reported in the
form m/z (intensity relative to base=100). High-resolution mass
spectra were acquired on a VG Instruments 70-S MS at the Chemistry
Department of Johns Hopkins University. The solvents used in
reactions were reagent grade. The solvents used for extraction and
chromatography were technical grade. All nonaqueous reactions were
performed in oven-dried glassware.
Abbreviations:
TfOH: trifluoromethanesulfonic acid; DBU:
1,8-Diazabicyclo[5.4.0]undec-7-ene
Synthesis of 4S/R-Cis-Itraconazole Stereoisomers
1-(2,4-Dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone
(2).sup.|33|
##STR00021##
To a suspension containing 13.8 g of 1H-1,2,4-triazole and 8.4 g of
NaHCO.sub.3 in 100 mL toluene, 22.4 g of
2-chloro-2',4'-dichlorophenylacetone (1) was added. The reaction
mixture was heated to 100.degree. C. for 3 h and then cooled to
-20.degree. C. overnight. The mixture was filtered to get the
yellowish solid. Then the collected solid was added to 200 mL water
and extracted with EtOAc (3.times.150 mL). The organic layer was
washed with brine and dried (MgSO.sub.4). The solvent was removed
and the solid was washed with EtOAc/hexanes (1:1, 100 mL) to get
ketone 2 (13.5 g 52.6%).
MP: 160-161.degree. C.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.24 (s, 1H), 7.97 (s,
1H), 7.64 (d, J=7.2 Hz, 1H), 7.50 (d, J=2 Hz, 1H), 7.38 (dd,
J.sub.1=2 Hz, J.sub.2=8.4 Hz, 1H), 5.64 (s, 2H)
MALDI-MS: 256.1 (M+H.sup.+), 258.1, 278.1 (M+Na.sup.+), 280.1.
((2S,4S)-2-((1H-1,2,4-Triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-diox-
olan-4-yl)methyl 4-methylbenzenesulfonate (3a)
##STR00022##
Under an atmosphere of argon, TfOH (1.5 mL, 16 mmol) was added to a
solution of (S)-1-tosyloxy-2,3-propanediol (1 g, 4 mmol) and ketone
2 (1 g, 3.9 mmol) in toluene (10 mL). Then the reaction mixture was
stirred at room temperature for 60 h. The reaction was quenched by
adding saturated aqueous NaHCO.sub.3 (25 mL), then extracted with
EtOAc (3.times.30 mL), washed with brine and dried over MgSO.sub.4.
The solvent was removed and the residue was re-dissolved in 2 mL
EtOAc. 4-Toluenesulfonic acid monohydrate (750 mg, 3.9 mmol) in
EtOAc (2 mL) was added dropwise to precipitate 3a preferentially as
a white solid. The mixture was stirred for 30 minutes and then
filtered to obtain salt 3a which was recrystallized from
acetonitrile to render it pure from the contaminating trans
diastereomer (1.44 g, 54.6%).
MP: 181-184.degree. C.
.sup.1H NMR (400 MHz, DMSO-d6): .delta. 8.33 (s, 1H), 7.82 (d,
J=8.4 Hz, 1H), 7.66 (d, J=2.4 Hz, 1H), 7.52 (d, J=8.0 Hz, 2H), 7.47
(d, J=8.0 Hz, 2H), 7.32-7.40 (m, 2H), 7.11 (d, J=8.0 Hz, 2H), 4.73
(s, 2H), 4.20-4.26 (m, 1H), 3.94 (dd, J.sub.1=4.0 Hz, J.sub.2=10.8
Hz, 1H), 3.74-3.82 (m, 2H), 3.63 (dd, J.sub.1=5.6 Hz, J.sub.2=8.8
Hz, 1H), 2.44 (s, 3H), 2.29 (s, 3H).
MALDI-MS: 484.1 (M+H.sup.+), 486.1, 506.1 (M+Na.sup.+), 508.1.
((2R,4R)-2-(1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxo-
lan-4-yl)methyl 4-methylbenzenesulfonate (3b).sup.|33|
##STR00023##
This compound was prepared from (R)-1-tosyloxy-2,3-propanediol and
ketone 2 following the procedure described above for making 3a.
MP: 184-186.degree. C.
To a solution of piperazine 4 (8 g, 41.6 mmol) and nitrobenzene 5
(6.3 g, 40.0 mmol) in DMSO (80 mL), potassium carbonate (6 g, 43.5
mmol) was added and the reaction mixture was heated at 160.degree.
C. overnight, and then cooled to room temperature. A red solid
crystallized which was filtered and washed with hot ethanol to get
piperazine 6. (12 g, 96% yield).
MP: 189-190.degree. C.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.16 (d, J=9.2 Hz, 4H),
6.87-6.97 (m, 4H), 3.80 (s, 3H), 3.58-3.60 (m, 4H), 3.22-3.24 (m,
4H).
MALDI-MS: 314.1 (M+H.sup.+), 336.1 (M+Na.sup.+).
4-(4-(4-Methoxyphenyl)piperazin-1-yl)-aniline (7).sup.|34|
##STR00024##
To a stirred suspension of compound 6 (9.5 g, 30 mmol) and
palladium catalyst (10% Pd on carbon 0.96 g) in methanol (100 mL),
ammonium formate (20 g, 317 mmol) was added slowly and the reaction
mixture was heated to reflux for 3 h. After cooling to room
temperature, the mixture was filtered and the filtrate was
concentrated. The residue was dissolved in water (50 mL) and
extracted with dichloromethane (3.times.40 mL). The organic layers
were combined, washed (brine), dried (MgS0.sub.4), and the solvent
was removed to get aniline 7 (5.1 g, 60% yield).
MP: 175-180.degree. C. (Dec.)
Phenyl 4-(4-(4-methoxyphenyl)piperazin-1-yl)phenylcarbamate
(8).sup.|40|
##STR00025##
To a solution of 7 (5.13 g, 1.8 mmol) and pyridine (12 mL, 145.2
mmol) in dichloromethane (50 mL), phenyl chloroformate (5 mL, 40.9
mmol) was added at 0.degree. C. The reaction mixture was stirred at
4.degree. C. overnight. Then water (100 mL) was added and the
mixture was stirred for 30 min. The precipitated white solid was
filtered and the solid was washed with dichloromethane (2.times.10
mL) and then dried under vacuum to obtain carbamate 8 (5.5 g, 75%
yield).
MP: 184-188.degree. C. (Dec).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.32-7.43 (m, 4H),
7.16-7.26 (m, 3H), 6.97 (d, J=8.4 Hz, 4H), 6.87 (d, J=8.8 Hz, 2H),
6.81 (s, 1H), 3.79 (s, 3H), 3.31 (s, 4H), 3.24 (s, 4H).
MALDI-MS: 404.1 (M+H.sup.+), 426.1 (M+Na.sup.+).
(.+-.)-N'-(2-butyl)formohydrazide (8a)
##STR00026##
This compound was prepared following a procedure reported for the
synthesis of closely related formohydrazide. To a solution of
2-butanone (1.5 g, 20.8 mmol) in tetrahydrofuran (20 mL) was added
formohydrazide (1.2 g, 20 mmol) and anhydrous sodium sulfate. The
flask was fitted with a Dean-Stark trap and heated to reflux for 2
hours. The reaction was cooled to room temperature and the solvent
was removed to get hydrazide 8a as a white solid which was used in
the reduction step directly without further purification.
The crude N'-(butan-2-ylidene)formohydrazide was dissolved in
methanol (20 mL). Sodium borohydride (600 mg, 15.8 mmol) was added
in portions at 0.degree. C. The reaction was stirred at room
temperature for 3.5 h. Then the solvent was removed and the residue
was dissolved in water (20 mL) and extracted with dichloromethane
(3.times.20 mL). The organic layers was combined, washed with brine
and dried over MgSO.sub.4. Then, the solvent was removed and the
residue was purified by flash chromatography (eluent:
EtOAc/hexanes=1:1) to obtain product 8a as a thick oil (1.67 g, 72%
yield over two steps).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.71-7.79 (m, 1H), 4.64
(br s, 1H), 3.77 (br s, 1H), 1.44-1.60 (m, 1H), 1.24-1.38 (m, 2H),
1.05 (dd, J.sub.1=2 Hz, J.sub.2=4 Hz, 3H), 0.92 (td, J.sub.1=3.2
Hz, J.sub.2=7.6 Hz, 3H).
(.+-.)-2-(sec-Butyl)-4-(4-(4-(4-methoxyphenyl)piperazin-1-yl)phenyl)-2H-1,-
2,4-triazol-3(4H)-one (9).sup.|40|
##STR00027##
To a solution containing 8 (2.1 g, 5 mmol) and
N'-sec-butylformohydrazide (8a) (0.72 g, 5.5 mmol) in toluene (25
mL), DBU (7.2 mg, 0.05 mmol) was added. The reaction mixture was
stirred and heated at 80.degree. C. for 7.5 h. followed by another
reflux for 8 h. After cooling to room temperature, the reaction
mixture was concentrated under reduced pressure, and methanol (8
mL) was added to the residue and the mixture was stirred at
4.degree. C. for 2 hours. Filtration of the mixture gave triazolone
9 as a white solid (1.85 g, 89% yield)
MP: 189-191.degree. C.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.62 (s, 1H), 7.43 (d,
J=8.8 Hz, 2H), 7.03 (d, J=8.8 Hz, 2H), 6.97 (d, J=9.2 Hz, 2H), 6.87
(d, J=9.2 Hz, 2H), 4.25-4.34 (m, 1H), 3.35-3.38 (m, 4H), 3.22-3.25
(m, 4H), 1.66-1.92 (m, 2H), 1.39 (d, J=6.8 Hz, 3H), 0.91 (t, J=7.2
Hz, 3H).
MALDI-MS: 408.3 (M+H.sup.+).
(.+-.)-2-sec-Butyl-4-(4-(4-(4-hydroxyphenyl)piperazin-1-yl)phenyl)-2H-1,2,-
4-triazol-3(4H)-one (21).sup.|40|
##STR00028##
Triazolone 9 (0.50 g, 1.2 mmol) was added to HBr (48%, 5 mL). The
reaction mixture was heated to 120.degree. C. and refluxed
overnight. The reaction mixture was cooled to room temperature,
while a pink solid precipitated. The solid was collected by
filtration, dissolved in methanol-water (1:1, 40 mL), saturated
aqueous NaHCO.sub.3 (20 mL) was added, and extracted with
chloroform (3.times.20 mL). The organic layer was washed with brine
and dried over MgSO.sub.4. The solvent was removed to obtain phenol
10 (0.48 g, 98% yield).
MP: 76-78.degree. C.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.63 (s, 1H), 7.39 (d,
J=8.8 Hz, 2H), 7.00 (d, J=9.2 Hz, 2H), 6.85 (d, J=8.8 Hz, 2H), 6.72
(d, J=8.8 Hz, 2H), 5.74 (br s, 1H), 4.26-4.35 (m, 1H), 3.32-3.40
(m, 4H), 3.18-3.25 (m, 4H), 1.65-1.91 (m, 2H), 1.41 (d, J=6.8 Hz,
3H), 0.91 (t, J=7.2 Hz, 3H).
MALDI-MS: 394.3 (M+H.sup.+), 416.3 (M+Na.sup.+).
4-(4-(4-(4-(((2S,4R)-2-((1H-1,2,4-Triazol-1-yl)methyl)-2-(2,4-dichlorophen-
yl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-sec-butyl-2H-
-1,2,4-triazol-3(4H)-one (11a).sup.|36|
##STR00029##
To a solution of tosylate 3a (260 mg, 0.39 mmol) and phenol 10 (140
mg, 0.36 mmol) in dry DMF (5 mL) potassium hydroxide (80 mg, 1.4
mmol) was added and the reaction mixture was heated at 50.degree.
C. overnight. After cooling to room temperature, the reaction
mixture was diluted with water (20 mL) and dichloromethane (20 mL),
and the organic phase was separated. The aqueous phase was
extracted with dichloromethane (2.times.20 mL). All the organic
layers were combined, washed with brine (20 mL), dried
(MgSO.sub.4), filtered, concentrated and purified by flash
chromatography on silica gel (eluent: from 1:1 EtOAc/hexanes to
neat EtOAc), affording (2S,4R)-itraconazole (11a) (113 mg, 45%
yield).
MP: 110-112.degree. C.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.22 (br s, 1H), 7.90
(br s, 1H), 7.62 (s, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.46 (d, J=2.0
Hz, 1H), 7.42 (d, J=9.2 Hz, 2H), 7.25 (dd, J=2.4 Hz, J.sub.2=8.4
Hz, 1H), 7.02 (d, J=8.8 Hz, 2H), 6.94 (d, J=8.8 Hz, 2H), 6.79 (d,
J=8.8 Hz, 2H), 4.80 (dd, J.sub.1=10.4 Hz, J.sub.2=34.0 Hz, 2H),
4.24-4.39 (m, 2H), 3.91 (dd, J.sub.1=6.8 Hz, J.sub.2=8.4 Hz, 1H),
3.76-3.82 (m, 2H), 3.47 (dd, J.sub.1=6.8 Hz, J.sub.2=9.6 Hz, 1H),
3.34-3.40 (m, 4H), 3.20-3.24 (m, 4H), 1.66-1.76 (m, 2H), 1.38 (d,
J=7.2 Hz, 3H), 0.89 (t, J=7.2 Hz, 3H).
.sup.13C-NMR (100 MHz, CDCl.sub.3), .delta.152.9, 152.3, 150.8,
146.2, 136.3, 134.3, 134.2, 133.4, 131.7, 129.8, 127.5, 126.2,
123.8, 118.7, 116.9, 115.5, 107.8, 74.9, 67.8, 67.7, 53.8, 52.9,
50.9, 49.4, 28.7, 19.5, 11.0.
MALDI-MS: 705.3 (M+H.sup.+), 707.3, 727.3 (M+Na.sup.+), 729.3.
HRMS for 11a: Calculated for C.sub.35H.sub.38C.sub.12N.sub.8O.sub.4
(M+H).sup.+: 705.2471, found 705.2457.
4-(4-(4-(4-(((2R,4S)-2-((1H-1,2,4-Triazol-1-yl)methyl)-2-(2,4-dichlorophen-
yl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-sec-butyl-2H-
-1,2,4-triazol-3(4H)-one (11b).sup.|36|
##STR00030##
This compound was prepared from tosylate 3b and phenol 10 following
the procedure described above for the case of the diastereomeric
itraconazole (11a) (11b, 44% yield).
MP: 109-111.degree. C.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.22 (s, 1H), 7.90 (s,
1H), 7.63 (s, 1H), 7.58 (d, J=8.8 Hz, 1H), 7.48 (d, J=2.0 Hz, 1H),
7.43 (d, J=8.8 Hz, 2H), 7.26 (dd, J.sub.1=2.0 Hz, J.sub.2=8.4 Hz,
1H), 7.03 (d, J=9.2 Hz, 2H), 6.94 (d, J==8.8 Hz, 2H), 6.80 (d,
J=8.8 Hz, 2H), 4.81 (dd, J.sub.1=14.4 Hz, J.sub.2=33.6 Hz, 2H),
4.24-4.39 (m, 2H), 3.92 (dd, J.sub.1=6.8 Hz, J.sub.2=8.4 Hz, 1H),
3.76-3.84 (m, 2H), 3.49 (dd, J.sub.1=6.0 Hz, J.sub.2=9.6 Hz, 1H),
3.34-3.40 (m, 4H), 3.20-3.24 (m, 4H), 1.66-1.76 (m, 2H), 1.39 (d,
J=7.2 Hz, 3H), 0.91 (t, J=7.2 Hz, 3H).
.sup.13C-NMR (CDCl.sub.3), .delta.152.8, 152.3, 150.8, 146.2,
136.3, 134.3, 134.2, 133.4, 131.7, 129.9, 127.5, 126.1, 123.8,
118.7, 116.9, 115.5, 107.8, 74.9, 67.8, 67.7, 53.8, 52.9, 50.8,
49.6, 28.7, 19.5, 11.0.
MALDI-MS: 705.4 (M+H.sup.+), 707.4, 727.4 (M+Na.sup.+), 729.4.
HRMS for 23: Calculated for C.sub.35H.sub.38Cl.sub.2N.sub.8O.sub.4
(M+H.sup.+): 705.2471, found 705.2456.
Synthesis of Azalanstat
4-(4-Chlorophenyl)butan-2-one (12).sup.|37|
##STR00031##
A mixture of 4-chlorobenzyl chloride (8.05 g, 55 mmol),
acetylacetone (5.20 mL, 50 mmol), K.sub.2CO.sub.3 (6.90 g, 50
mmol), and anhydrous ethanol (50 mL) was refluxed for 16 h. The
reaction mixture was cooled to room temperature and the solvent was
removed. Water (100 mL) was added to the residue and the mixture
was extracted with Et.sub.2O (3.times.50 mL). The combined organic
layers were washed with brine (50 mL) and dried over
Na.sub.2SO.sub.4 and concentrated to dryness. The resulting yellow
oil was distilled under reduced pressure (0.1 Torr) and the
fraction that distilled at 110.degree. C. was collected as a clear,
colorless oil (6.15 g, 67% yield).
TLC (3:7 EtOAc/hexanes): R.sub.f 0.485.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.7.26-7.20 (m, 2H),
7.11-7.08 (m, 2H), 2.84 (t, J=8.0 Hz, 2H), 2.72 (t, J=8.0 Hz, 2H),
2.12 (s, 3H).
1-Bromo-4-(4-chlorophenyl)butan-2-one (13)
##STR00032##
This compound was prepared following a procedure reported for
making 1-bromo-4-phenyl-butan-2-one. A freshly prepared solution of
bromine (910 .mu.L, 17.74 mmol) in MeOH (15 mL) was added dropwise
during 1 h 20 min to a stirred solution of benzylacetone (3.0 g,
16.42 mmol) in MeOH (15 mL) at 7-10.degree. C. An exothermic
reaction took place and to maintain the temperature at 7-10.degree.
C., the flask was immersed in an ice-water bath when necessary.
Once the orange-red color of bromine disappeared, water (25 mL) was
added and the mixture was stirred overnight. The organic layer was
separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2
(2.times.30 mL). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure.
The oily residue was dissolved in hexanes (30 mL) and the soln. was
refrigerated overnight (-10.degree. C.). Fine needles precipitated
and they were filtered off, washed with cold hexanes, and dried
under vacuum at room temperature to afford butanone 13 (2.76 g,
64%).
MP: 51.degree. C.; TLC (1:4 EtOAc/hexanes): R.sub.f 0.455.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.7.26 (d, J=6.86 Hz, 2H),
7.13 (d, J=6.86 Hz, 2H), 3.83 (s, 2H), 3.00-2.84 (complex set of
triplets, 4H).
MALDI-MS: m/z 262.2 (M+H.sup.+), 286.5 (M+Na.sup.+).
1-(4-(4-Chloro)phenyl-2-oxobut-1-yl)imidazole (14).sup.|39|
##STR00033##
Butanone 13 (2.76 g, 10.55 mmol) was added portionwise over half an
hour to a stirred suspension of imidazole (3.6 g, 53 mmol) in DMF
(20 ml) at 0.degree. C. and the mixture was stirred overnight at
ambient temperature. The resulting solution was poured into water
(50 mL) and the mixture was extracted with CH.sub.2Cl.sub.2
(3.times.25 mL). The organic layers were pooled and concentrated to
dryness. Chromatography of the crude product on silica gel eluting
with 5% methanol in CH.sub.2Cl.sub.2 gave imidazole derivative 14
(2.46 g, 94% yield). The hydrochloride salt crystallized from
acetone/methanol (1:1) melted at 173.degree. C.
TLC (5% MeOH/CH.sub.2Cl.sub.2): R.sub.f 0.351 (tailing).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.01 (s, 1H), 7.57 (d,
J=4.04 Hz, 1H), 7.28-7.22 (m, 2H), 7.13-7.06 (m, 2H), 6.83 (d,
J=4.04 Hz, 1H), 4.64 (s, 2H), 2.93-2.86 (m, 2H), 2.75-2.69 (m,
2H).
MALDI-MS: m/z 249.2 (MH.sup.+), 271.2 (M+Na.sup.+).
(2SR,4S)-2-(1-Imidazolylmethyl)-2-(2-(4-chlorophenyl)ethyl)-4-(4-methylben-
zenesulfonyloxy-methyl)-1,3-dioxalane (15a and 15b).sup.|40|
##STR00034##
In a two-necked flask provided with a Dean-Stark trap,
methanesulfonic acid (780 .mu.L) was added slowly to a solution of
butanone 14 (1.0 g, 4.02 mmol) and
(25)-2,3-dihydroxyprop-1-yl-(4-methyl)-benzenesulfonate (1.123 g,
4.56 mmol) in dichloromethane (25 mL) at room temperature. After
completing the addition, the solution was heated to reflux for 24
h, cooled to room temperature, and slowly poured into a mixture of
ice (20 g), water (40 mL), potassium carbonate (10 g), and
dichloromethane (60 mL). The organic layer was separated, and the
aqueous phase was extracted with dichloromethane (3.times.25 mL).
The combined dichloromethane layers were dried over
Na.sub.2SO.sub.4 and filtered, and the filtrate was evaporated
under reduced pressure. The residue was purified by column
chromatography on silica gel eluting with chloroform), and the
diastereomeric mixture of 15a and 15b was obtained as a beige solid
(1.38 g, 72% yield).
TLC (5% MeOH/CH2Cl2): Rf 0.44 (tailing).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.02 (s, 1H), 7.80-7.77
(m, 2H), 7.42 (d, J=4.04 Hz, 1H), 7.24-7.22 (m, 3H), 7.18-7.13 (m,
3H), 7.01 (d, J=4.04 Hz, 1H), 4.37-4.32 (m, 1H), 4.15-4.03 (m, 3H),
3.75-3.72 (m, 2H), 3.27-3.23 (m, 1H), 2.92-2.88 (2 s, s, 3H),
2.67-2.64 (m, 2H), 2.43-2.41 (m, 1H), 1.94-1.91 (m, 2H).
MALDI-MS: m/z 477.5 (MH.sup.+), 500.3 (M+Na.sup.+).
(2S/R,4S)-Azalanstat (16a and 16b)
##STR00035##
Under a N.sub.2 atmosphere, a mixture of tosylate 15a and 15b (450
mg, 0.94 mmol), 4-aminothiophenol (247 mg, 1.97 mmol), and
K.sub.2CO.sub.3 (260 mg, 1.88 mmol) in acetone was heated to reflux
temperature with stirring for 5 hours. The solids were removed by
filtration, and washed with hot acetone and then with hot EtOAc.
The filtrate was concentrated, and the residue was purified by
flash chromatography on silica gel (EtOAc) to give a mixture of 16a
and 16b (280 mg, 69% yield as a 2:3 mixture of 2S,4S i.e. 16a and
2R,4S i.e. 16b). The beige solid was then suspended in warm 1:3
Et.sub.2O/CH.sub.2Cl.sub.2 (25 mL) and allowed to stand overnight
in the freezer (-20.degree. C.). Beige crystals were collected by
filtration (98.6 mg, 16a (4S,2S), MP: 151.degree. C.) and the
solution was concentrated to obtain the trans diastereomer (152.3
mg, 16b (4S,2R)).
TLC (5% MeOH/CH.sub.2Cl.sub.2): R.sub.f0.32 (16a), 0.19 (16b).
.sup.1H NMR of 16a/16b (400 MHz, Acetone-d6): .delta. 8.45 (d,
J=9.8 Hz, 1H), 7.62 (t, J=6.7 Hz, 2H), 7.54 (apparent d, J=7.7 Hz,
4H), 7.45 (t, J=6.7 Hz, 2H), 6.34 (apparent t, J=1.4 Hz, 2H),
4.69-4.63 (m, 1H), 4.57-4.54 (m, 1H), 4.48-4.38 (m, 1H), 4.23 (d,
J=2.7 Hz) and 4.16 (d, J=2.9 Hz) together 1H, 3.73-3.66 (m, 1H),
3.58-3.45 (m, 2H), 3.36-3.24 (m, 1H), 3.16 (d, J=2.9 Hz) and 3.02
(dd, J=3.3 and 0.9 Hz) together 1H, 2.74-2.70 (m, 1H), 1.84 (d,
J=3.3 Hz) and 1.62 (d, J=1.8 Hz) together 1H.
MALDI-MS: m/z 430.3 (MH.sup.+).
EXAMPLE 2
4S/R-Cis-Itraconazole and Azalanstat Biological Studies
Cell Culture.
HUVEC were purchased from Cambrex Biosciences (Walkersvilie, Md.)
and maintained in EGM-2 media (Cambrex) which contains VEGF, bFGF,
and EGF. LPDS were purchased from Intracell. BAEC and Hela cells
were maintained in DMEM media containing 10% FBS. Jurkat cells were
maintained in RPMI media containing 10% FBS. In a typical
experiment 5,000-10,000 cells/well in 0.2 mL EGM-2 media were
allowed to adhere for 8 h and then incubated with drug for 36 h.
Cells were pulsed with 1 .mu.Ci [.sup.3H]-thymidine for 8 h (MP
Biomedicals, 6.7 Ci/mmol), and harvested using trypsin onto glass
fiber filters (Wallac, Turku, Finland). The readout was performed
on a Perkin Elmer MicroBeta plate reader. Cells used were under
five passages.
Human foreskin fibroblast (HFF) cells were cultured at passage 2 in
DMEM low glucose, 10% fetal bovine serum, and 1%
penicillin-streptomycin. Experiments were performed with 2,500
cells/well in 0.2 mL and incubated for 96 h with drug. Plates
containing cells were washed once with PBS, incubated with 1 .mu.M
Calcein-AM (Molecular Probes) in PBS for 4 h, and read in a
fluorescent plate reader. IC.sub.50 values were determined using
four-parameter logarithmic analysis with GraphPad Prism and are
presented as mean.+-.s.e.m. for triplicate experiments.
Endothelial cells form the inner lining of all blood vessels and
constitute an essential part of new as well as pre-existing blood
vessels. An endothelial cell proliferation assay was utilized to
screen our clinical drug library. The screen was carried out in
96-well plates with each drug at a final concentration of 10 .mu.M.
Thus, HUVEC were incubated with drugs for 36 h and proliferation
was measured by following incorporation of [3H]-thymidine for the
final 8 h. The preliminary screen identified 210 existing drugs
with at least 50% inhibition at 10 .mu.M, which belong to multiple
drug classes (FIG. 1a).
Itraconazole displayed quite potent and selective inhibitory
activity toward endothelial cells compared to other cell types
tested. For example, itraconazole has little effect on the
proliferation of human foreskin fibroblasts (HFF) with an IC50 over
20 .mu.M in comparison to HUVEC (IC50=0.16 .mu.M) (FIG. 1b). While
it potently inhibited the proliferation of bovine aortic
endothelial cells (BAEC), it is much less effective against Jurkat
T cells or HeLa cells.
To delineate the mechanism of inhibition of endothelial cell
proliferation by itraconazole, the effect on the cell cycle
progression of HUVEC by fixing and staining cells with propidium
iodide followed by FACS analysis was examined. The 4S-cis
diastereomer potently inhibits HUVEC cell cycle progression at the
G1/S transition (FIG. 1c). Treatment of HUVEC with racemic
itraconazole also led to an increase of cells in the G1 phase of
the cell cycle and a corresponding decrease in cells in the S phase
(data not shown). These results indicate that itraconazole inhibits
HUVEC proliferation by blocking cell cycle progression in the G1
phase.
Two complementary approaches were taken to assess the relevance of
14DM in the inhibition of endothelial cell proliferation by
itraconazole. First, a known potent inhibitor of human 14DM,
azalanstat, was prepared and its effect on endothelial cells was
compared with that of itraconazole. Similar to itraconazole,
azalanstat also blocked the cell cycle progression of HUVEC (FIG.
2) and BAEC in the G1 phase of the cell cycle (FIG. 3 and Table 1),
suggesting that 14DM is required for endothelial cell
proliferation.
TABLE-US-00001 TABLE 1 Effects of itraconazole and azalanstat on
the cell cycle progression of BAEC*. Control Cholesterol G1 S G2/M
G1 S G2/M Control 66.4 18.7 14.2 65.5 22.1 11.8 Itraconazole 77.0
10.5 12.0 66.2 21.2 12.0 Azalanstat 74.5 16.8 8.8 70.2 19.4 10.0
*Values represent the percentage of cells in a given phase of the
cell cycle.
A hallmark of inhibitors of 14DM is that their potencies are
dependent on the levels of cholesterol in cell culture medium.
Thus, the potencies of both azalanstat and itraconazole in cell
culture media either containing or lacking cholesterol was
determined.
Azalanstat displayed higher potency toward endothelial cells in the
absence of cholesterol (IC.sub.50=0.31 .mu.M) than in its presence
(IC.sub.50=1.2 .mu.M) (FIG. 2a). Similarly, the inhibition of
endothelial cells by itraconazole was also sensitive to
cholesterol, being less potent when cholesterol is present
(IC.sub.50=0.044 .mu.M vs. 0.23 .mu.M) (FIG. 2b).
In contrast, an inhibitor of angiogenesis with unrelated mechanism
of action, TNP-470, that works by inhibiting the type 2 methionine
aminopeptidase, inhibited endothelial cell proliferation with
roughly equal potency in the absence and presence of cholesterol
(FIG. 2c).
Together, these observations suggest that itraconazole works at
least in part by inhibiting cholesterol biosynthesis.
The second approach was to knockdown the expression of human 14DM
in HUVEC and determine the effect on cell proliferation. Thus,
three different shRNAs targeting the coding region of human 14DM
mRNA were transiently expressed in 293T cells along with the
expression plasmid for human 14DM with an C-terminal c-Myc tag.
One of the constructs, pSSII-sih14DM, dramatically blocked the
expression of ectopically expressed protein (FIG. 3a). The
expression cassette for this shRNA was then moved to the lentiviral
vector, pFUP2, and the resulting lentiviruses were generated and
used to transduce HUVEC.
As shown in FIG. 3b, the human 14DM lentiviral shRNA blocked the
expression of endogenous 14DM expression, as judged by RT-PCR about
3 days after viral transduction.
The transduced cells were allowed to grow and their proliferation
in the absence and presence of varying concentrations of
itraconazole was determined at days 7 and 10 post-transduction,
respectively. HUVEC transducted with human 14DM shRNA proliferate
more slowly than those transduced with the control viruses, as
judged by the amounts of [3H]-thymidine incorporated at Day 7 (FIG.
3c).
Together, these results demonstrate that 14DM is essential for
endodielial cell growth and suggest that human 14DM may serve as a
novel target for developing angiogenesis inhibitors.
To determine whether itraconazole inhibited angiogenesis in vivo,
itraconazole was tested in a mouse Matrigel model. In humans,
itraconazole is administered intravenously at a dose of 105
mg/m.sup.2 twice daily. Mice were thus treated with a comparable
dose of itraconazole (112.5 mg/m2 or 37.5 mg/kg, i.p. once
daily).
Female athymic nude 5-week old, 25-30 g mice were purchased from NO
and treated in accordance with standard procedures. In all animal
experiments the i.v. formulation of itraconazole was obtained from
the Johns Hopkins Hospital Pharmacy. Control mice were treated with
vehicle (40% hydroxypropyl-.beta.-cyclodextrin, 2.5% propylene
glycol, pH 4.5). Mice were pretreated for three days and then
implanted subcutaneously with 0.5 mL of Matrigel (BD Biosciences)
containing 100 ng/mL VEGF and 150 ng/mL bFGF. Drug treatment was
continued daily for 10 days, mice were sacrificed, and plugs were
harvested, fixed in neutral buffered formalin, and processed for
histology using MAS-trichrome staining. A cross section of the
entire Matrigel plug was photographed at 100.times. and
erythrocyte-filled blood vessels were counted per field in a
blinded manner. P-values comparing itraconazole versus vehicle
treated mice were determined using the two-tailed Student's T-test;
and the data are presented as mean.+-.s.e.m.
A significant decrease in angiogenesis was observed both
macroscopically, as judged by the red color of the isolated
Matrigel plugs (FIG. 4a), and microscopically upon staining thin
sections for new blood vessels (FIG. 4b) in animals treated with
itraconazole. Overall, there was a 67.5% decrease in new blood
vessel formation in itraconazole-treated mice compared to
vehicle-treated controls (FIG. 3c), indicating itraconazole is
capable of suppressing angiogenesis in vivo.
EXAMPLE 3
Itraconazole Stereoisomers
General Experimental
Reactions were carried out in oven dried glassware. All reagents
were purchased from commercial sources and were used without
further purification unless noted. Unless stated otherwise, all
reactions were carried out under a positive pressure of argon and
were monitored by Merck precoated silica gel 60F-254 plates and
were visualized using 254 nm UV light. Column chromatography was
performed on silica gel (200-400 mesh, Merck). The ratio between
silica gel and crude product ranged from 100 to 50:1 (w/w). NMR
data were collected on a Varian Unity-400 (400 MHz 1H, 100 MHz 13C)
machine in the Department of Pharmacology and Molecular Sciences,
the Johns Hopkins University. 1H NMR spectra were obtained in
deuteriochloroform (CDCl.sub.3) with either tetramethylsilane (TMS,
.delta.=0.00 for .sup.1H) or chloroform (CHCl.sub.3, .delta.=7.27
for .sup.1H) as an internal reference. .sup.13C NMR spectra were
proton decoupled and were in CDCl.sub.3 with either TMS
(.delta.=0.0 for .sup.13C) or CHCl.sub.3 (.delta.=77.0 for
.sup.13C) as an internal reference. Chemical shifts are reported in
ppm (.delta.). Data are presented in the form: chemical shift
(multiplicity, coupling constants, and integration). .sup.1H data
are reported as though they were first order. The errors between
the coupling constants for two coupled protons were less than 0.5
Hz, and the average number was reported. The low-resolution mass
spectra were obtained on a Voyager DE-STR, MALDI-TOF instrument at
the AB Mass Spectrometry/Proteomics Facility at the Johns Hopkins
University. The MALDI-samples were prepared by mixing droplets of
the sample solutions in chloroform or methanol and
2,5-dihydroxybenzoic acid solution in acetone, where the latter
served as the matrix. The high-resolution mass spectra were
acquired on an ABI Voyager-DE STR instrument in the Department of
Chemistry at the Texas A&M University. The ionization method is
MALDI by using THAP (2,4,6-trihydroxyacetophenone) as the matrix.
Optical rotations were measured on a Jasco P-1010 polarimeter at
the sodium D line (589 nm) in the Department of Chemistry, the
Johns Hopkins University. Optical rotations were measured at
22.+-.2.degree. C. Optical rotations are in units of
degmL(dmg).sup.-1.
Synthesis and Isolation of Itraconazole Stereosiomers
Trans-(2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-4-tos-
yloxymethyl-1,3-dioxolane (22c) or
trans-(2S,4R)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-4-to-
syloxymethyl-1,3-dioxolane (22d)
After the salt formation with toluenesulfonic acid, the majority of
tosylate salts of 22c or 22d were remained in the ethyl acetate
solution, which was neutralized by washing with a saturated aqueous
K.sub.2CO.sub.3 solution. The aqueous layer was then extracted with
CH.sub.2Cl.sub.2 several times. The combined organic solution was
dried (Na.sub.2SO.sub.4), filtered, and concentrated to yield the
crude product, which was purified by column chromatography
(50:1.fwdarw.5:1 CH.sub.2Cl.sub.2-acetone) to afford 22c or 22d as
a yellowish oil: Rf 0.33 (5:1, CH.sub.2Cl.sub.2-acetone); .sup.1H
NMR (400 MHz, CDCl.sub.3, .delta.H) 8.11 (s, 1H), 7.85 (s, 1H),
7.60 (d, J=8.1 Hz, 2H), 7.49-7.22 (m, 4H), 7.09 (dd, J=8.5, 1.4 Hz,
1H), 4.73-4.60 (m, 2H), 4.14-3.98 (m, 1H), 3.94-3.80 (m, 3H), 3.59
(t, J=7.8 Hz, 1H), 2.43 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3,
.delta..sub.C) 151.69, 145.53, 144.96, 136.14, 134.54, 133.00,
132.32, 131.33, 130.15, 129.40, 128.03, 127.38, 108.28, 75.01,
67.69, 67.07, 54.37, 21.92.
MALDI-MS: 484.1 (M+H.sup.+), 506.1 (M+Na.sup.+).
Protocol for the Preparation of trans-Itraconazole 23e-23h
To a solution of 20a or 20b (22.6 mg, 0.057 mmol) in DMSO was added
NaH (10.2 mg of a 60% dispersion in mineral oil, 0.26 mmol). After
the mixture was stirred at 50.degree. C. under argon for 1 h, a
solution of 22c or 22d (30.0 mg, 0.062 mmol) in DMSO was added
dropwise. After the addition, the temperature was increased to
90.degree. C., and the solution was stirred under argon for another
3 h. The reaction was then quenched by the addition of a 50%
aqueous NaCl solution, and the resulting mixture was extracted with
CH.sub.2Cl.sub.2. The organic fractions were dried
(Na.sub.2SO.sub.4), filtered, and concentrated under vacuum to
yield the crude product, which was purified by column
chromatography (1:1 hexanes-EtOAc.fwdarw.neat EtOAc) to afford
trans-itraconazole (27.9 mg, 69%) as a yellowish oil, which could
be further purified by a second column (neat
CH.sub.2Cl.sub.2.fwdarw.50:1 CH.sub.2Cl.sub.2--CH.sub.3OH) if
necessary: Rf 0.56 (EtOAc); .sup.1H NMR (400 MHz, CDCl.sub.3,
.delta..sub.H) 8.23 (br s, 1H), 7.93 (br s, 1H), 7.65-7.55 (m, 2H),
7.49-7.38 (m, 3H), 7.19 (dd, J=8.5, 2.1 Hz, .sup.1H), 7.13-6.85 (m,
4H), 6.68 (t, J=15.2 Hz, 2H), 4.76 (q, J=14.6 Hz, 2H), 4.36-4.15
(m, 2H), 4.00 (dd, J=8.2, 6.4 Hz, 1H), 3.89 (dd, J=10.0, 4.5 Hz,
1H), 3.80 (dd, J=13.9, 6.6 Hz, 2H), 3.20-3.57 (m, 8H), 1.94-1.77
(m, 1H), 1.78-1.64 (m, 1H), 1.39 (d, J=6.7 Hz, 3H), 0.89 (t, J=7.4
Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3, .delta..sub.C) 152.22,
136.03, 135.23, 134.09, 133.21, 131.37, 129.69, 127.30, 123.79,
119.12, 117.12, 115.48, 108.05, 76.25, 67.79, 67.60, 54.66, 52.90,
51.30, 49.12, 28.66, 19.50, 11.03.
MALDI-MS: 705.3 (M+H.sup.+), 727.3 (M+Na.sup.+).
HPLC Analysis of Itraconazole Diastereomers 23a-23h
HPLC: JASCO PU-2089S Plus quaternary pump system Column:
CHIRALPAK.RTM. AS-RH (2.1.times.150 mm), 5 .mu.m Detector: MD-2010
Plus PDA Conditions
Itraconazole diastereomers were analyzed by HLPC. Briefly, 10 .mu.L
sample of each itraconazole diastereomer as .about.1 mM solution
inchloform was injected into a 7725i Rheodyne injection module. A
mixture of acetonitrile and 5 mM aqueous ammonium acetate was used
as the mobile phase with the flow rate of 0.2 mL/min. Initially,
mobile phase composition was 70% 5 mM aqueous ammonium acetate and
30% acetonitrile, but the gradient was ramped up to 70%
acetonitrile and 30% 5 mM aqueous ammonium acetate over 46 min
starting at the 4.sup.th min. In the next 5 min, eluent was changed
to 20% 5 mM aqueous and 80% acetonitrile to wash off the column,
and then it was switched to the original composition to get ready
for the next injection cycle.
Biological Methods
Pooled HUVEC and EGM-2 bullet kits were purchased from Lonza.
Powdered RPMI 1640 media with L-glutamine and without sodium
bicarbonate was purchased from Gibco. Amino acids, uracil, and agar
were purchased from Sigma and morpholinepropanesulfonic acid (MOPS)
was purchased from Fisher. Dimethyl Sulfoxide (DMSO) was purchased
from J. T. Baker. C. glabrata strains BG1 (J. Infect. Dis. 1996,
173, 425-431) and BG2 (Genetics 1999, 151, 979-987), C. neoformans
H99 (Am. J. Pathol. 1980, 101, 177-193), S. cerevisiae BY4741
(MATa, ura3, his3, leu2, met15), and C. albicans (ATCC 10261) were
used.
Cell Culture
HUVEC between passage 3 and 8 were grown in EGM-2 bullet kit media
at 37.degree. C. in a humidified environment with 5% CO.sub.2
present. Fungi were maintained on YES (5 g/L yeast extract, 30 g/L
glucose) agar (2%) plates supplemented with 225 mg/L adenine,
histidine, leucine, uracil, and lysine at 30.degree. C.
HUVEC Proliferation Assay
HUVEC were plated in a 96-well plate at a density of 2000
cells/well 199 .mu.L media. After recovering overnight, cells were
incubated for 24 hours with drugs added from 200.times.DMSO stocks.
Next, cells were incubated with 0.9 .mu.Ci of [.sup.3H]-thymidine
for 6 h, washed once with PBS, trypsinized, and transferred to
filtermats (Wallac) using a Mach III M Harvester 96 (Tomtec). The
filtermats were then dried overnight. Using a 1450 Microbeta
apparatus (Wallac), the amount of [.sup.3H] at each position on the
filtermat was determined by scintillation counting. Counts from
vehicle only treated controls were used to normalize for maximum
proliferation. Experiments were conducted in triplicate with
multiple technical replicates for each data point. A four parameter
logistic regression was used to determine IC.sub.50 values
(GraphPad Prism [v4.03]).
Fungal Susceptibility Assays
Fungi were grown overnight in 5 mL YES supplemented 225 mg/L
adenine, histidine, leucine, uracil, and lysine (supplemented YES)
at 30.degree. C. with shaking. Cultures were diluted to 0.01
OD.sub.600 in RPMI (with L-glutamine, without sodium bicarbonate)
which was supplemented with 2% glucose, 225 mg/L adenine,
histidine, methionine, leucine, uracil, and lysine and buffered
with 165 mM MOPS to 7.0 (complete RPMI). This solution was then
further diluted 190-fold in complete RPMI in 96-well plates
containing test compounds in a final volume of 150 .mu.L. When
practical, the intermediate RPMI dilution was bypassed and the
overnight cultures were directly diluted to the appropriate
OD.sub.600 in the RPMI solution that was added to the 96-well
plates. Two-fold serial dilutions of the compounds were tested
starting at 4 ug/mL with each well containing 0.125% vehicle
(DMSO). The plates were incubated in a humidified environment at
30.degree. C. with shaking for 30 hours except for experiments with
C. neoformans which was incubated for 54 h. Abs.sub.600 was
measured using a plate reader (Bio-Tek Synergy HT) after vigorous
shaking for 45 seconds to resuspend the cells. The background
absorbance from noninoculated wells was subtracted and the
absorbance of test wells was divided by that of wells treated with
vehicle alone. The percent of remaining growth for each drug
concentration was averaged over 2-3 independent experiments, each
with two technical replicates, and the minimum concentration giving
80% inhibition (MIC.sub.80) was determined.
After the quality of all stereoisomers was confirmed, the potency
of each stereoisomer against HUVEC proliferation and fungal growth
was determined (FIG. 7). HUVEC were incubated with drug or vehicle
alone for 24 hours and then pulsed for 6 hours with
[.sup.3H]-thymidine, the incorporation of which was taken as a
readout of cell proliferation. Inhibition of fungal growth was
assayed by incubating five yeast strains with 2-fold serial
dilutions of each stereoisomer for 30-60 hours depending on the
strain, and then measuring the OD.sub.600 of the culture to
quantitate growth. The minimum concentration capable of inhibiting
growth by 80% (MIC.sub.80) was determined.
The influence of stereochemistry on the inhibition of HUVEC
proliferation by itraconazole was minor. The difference in potency
between 23a and 23f, the most and least potent stereoisomers,
respectively, was only slightly greater than 4-fold. The most
relevant stereochemical determinant of potency in HUVEC was the
configuration of the dioxolane ring, with the cis-diastereomers
exhibiting higher potency than the trans series by several fold.
The cis-4R diastereomer was found to be slightly more potent than
the cis-4S isomer, with correct stereochemical centers having been
assigned. In contrast to HUVEC, the potency of itraconazole against
fungal proliferation was highly influenced by stereochemistry (FIG.
7). A difference in potency of up to 32-fold was observed between
stereoisomers in one fungal strain. In four out of five strains
tested the least potent stereoisomers by a margin of at least 4- to
32-fold were two of the trans isomers, 23g and 23h. On the other
hand, the other trans pair 23e and 23f were about as potent as the
cis diastereomers (23a through 23d). The exception was C.
neoformans in which 23e and 23f were 2-fold less potent than 23g
and 23h and 32-fold less potent than the best inhibitor.
All references cited herein, whether in print, electronic, computer
readable storage media or other form, are expressly incorporated by
reference in their entirety, including but not limited to,
abstracts, articles, journals, publications, texts, treatises,
technical data sheets, internet web sites, databases, patents,
patent applications, and patent publications.
Certain compounds of the present invention may exist in particular
geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such enriched isomers, as well as racemic mixtures thereof, are
intended to be included in this invention.
Although the invention has been described with reference to the
above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *